Anti-vista antibodies and fragments

ABSTRACT

The present invention relates to novel antibodies and fragments that bind to a V-domain Ig Suppressor of T cell Activation (VISTA), and methods of making and using same. Methods of use include methods of treatment of cancer, including leukemias, lymphomas, solid tumors and melanomas.

RELATED APPLICATION(S)

This application claims the benefit of U.S. Provisional Application No.61/920,695, filed on Dec. 24, 2013 and U.S. Provisional Application No.62/085,086, filed on Nov. 26, 2014. The entire teachings of the aboveapplications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

The expression of negative immune checkpoint regulators by cancer cellsor immune cells in the tumor microenvironment can suppress the host'simmune response against the tumor. To effectively combat the cancer, itis desirable to block tumor-mediated suppression of the host immuneresponse. Accordingly, there is a need for new and effective therapeuticagents that inhibit negative immune checkpoint regulators in the tumormicroenvironment that suppress anti-tumor immune responses.

SUMMARY OF THE INVENTION

The present invention relates to antibodies and antibody fragmentscomprising an antigen binding region that binds to a V-domain IgSuppressor of T cell Activation (VISTA). VISTA is a checkpoint regulatorthat negatively suppresses immune responses. See Wang et al., “VISTA, anovel mouse Ig superfamily ligand that negatively regulates T cellresponses,” J. Exp. Med., 208(3) 577-92 (2011). It is expressed onnormal human neutrophils, monocytes and T cells subsets. In addition,cynomolgus monkey cells express VISTA in a similar pattern to normalhuman cells. VISTA is also expressed in the peripheral blood cells ofcancer patients.

The binding of the antibody or antibody fragment to VISTA modulates orenhances an immune response. The antibody fragment can include, forexample, a Fab, F(ab′)₂, or scFv antibody fragment. The antibody orantibody fragment can comprise an antibody constant region. The antibodyor antibody fragment can bind to VISTA that is expressed on ahematopoietic cell, for example, a myeloid cell and/or a lymphocyte, amonocyte or a neutrophil, a T cell, a natural killer (NK) cell, anatural killer T (NKT) cell, a tumor cell, and/or in the tumormicroenvironment (TME). The tumor microenvironment is the cellularenvironment of the tumor. It can include surrounding immune cells,fibroblasts, blood vessels, other cells, signaling molecules, and theextracellular matrix.

The antibody or antibody fragment can comprise one or more heavy chaincomplementary determining regions (CDRs) and/or one or more light chainCDRs, including one or more CDRs (e.g., all three heavy chain CDRs, all3 light chain CDRs, or all 6 CDRs) of any of the anti-VISTA antibodiesdescribed herein, including the antibodies designated VSTB112 (S2),VSTB116 (S5), VSTB95 (S16), VSTB50 (S41), VSTB53 (S43) and VSTB60 (S47).In some embodiments, the antibodies or fragments thereof are selectedfrom the group consisting of VSTB112, VSTB95, VSTB116, VSTB50, VSTB53and VSTB60. In one embodiment, the antibody or fragment comprises one ormore of the heavy chain CDRs and one or more light chain CDRs of any ofthe anti-VISTA antibodies described herein. In some embodiments, theantibody or antibody fragment can further comprise at least one heavychain and at least one light chain of any of the anti-VISTA antibodiesdescribed herein. In some embodiments, the antibody or antibody fragmentcomprises at least one heavy chain comprising the heavy chain variableregion sequence, and/or at least one light chain comprising the lightchain variable region sequence. In some embodiments, the antibodycomprises a human framework region. In some embodiments, the antibody isa whole antibody. In some embodiments, the fragment is an anti-VISTAbinding member. In some embodiments, the heavy chain CDRs of theantibody are represented in SEQ ID NOs:1, 2, and 3 and the light chainCDRs are represented in SEQ ID NOs: 4, 5, and 6. In some embodiments,the heavy chain and light chain variable region amino acid sequences arerepresented in SEQ ID NOS: 7 and 8, respectively.

The invention also encompasses anti-VISTA antibodies which aresubstantially similar to antibodies described herein. For example, inone embodiment, the antibody or fragment comprises an antibody VH domaincomprising a VH CDR1 having an amino acid sequence that is substantiallysimilar to SEQ ID NO:1, a VH CDR2 having an amino acid sequence that issubstantially similar to SEQ ID NO:2 and a VH CDR3 having an amino acidsequence that is substantially similar to SEQ ID NO:3, and which furthercomprises an antibody VL domain comprising a VL CDR1 having an aminoacid sequence that is substantially similar to SEQ ID NO:4, a VL CDR2having an amino acid sequence that is substantially similar to SEQ IDNO:5 and a VL CDR3 having an amino acid sequence that is substantiallysimilar to SEQ ID NO:6.

The invention also relates to anti-VISTA antibodies that competitivelyinhibit, or compete for binding to, the anti-VISTA antibodies describedherein.

In some embodiments, the anti-VISTA antibodies are part of a conjugate,for example, a conjugate that comprises a cytotoxic molecule or anotheragent described herein. Such molecules are well-known in the art.

In some embodiments, the antibody or antibody fragment is a monoclonalantibody. In some embodiments, the antibody is a chimeric, humanized orhuman antibody. In some embodiments, the antibody or antibody fragmentcomprises a human constant region. In some embodiments, the antibody orantibody fragment is specific for an epitope of VISTA, e.g., within theamino acid sequence SEQ ID NO: 9. In some embodiments, the antibody orantibody fragment binds to an epitope of VISTA with an affinity of atleast 1×10⁻⁷ liter/mole, for example, at least 1×10⁻⁸ liter/mole, forexample, at least 1×10⁻⁹ liter/mole.

In some embodiments, the modulation of the immune response comprises anincrease in CD45+ leukocytes, CD4+ T cells, and/or CD8+ T cells. In someembodiments, the modulation of the immune response comprises enhancedproduction of (e.g., T-cell) cytokines (e.g., IFNγ, IL-10, TNFα, IL-17),enhanced T-cell response, and/or modulated Foxp3 expression.

The present invention also relates to compositions comprising anantibody or antibody fragment described herein (e.g., an anti-VISTAantibody) and a pharmaceutically acceptable carrier, diluent, orexcipient. For example, the composition can comprise a VISTA antagonistcomprising an antibody or antibody fragment thereof comprising anantigen binding region that binds to VISTA, and a vaccine (such as aviral vector vaccine, bacterial vaccine, DNA vaccine, RNA vaccine,peptide vaccine). In some embodiments, the composition is apharmaceutical composition and binding of the antibody or antibodyfragment to VISTA modulates or enhances an immune response.

The invention also relates to methods for treating or preventing cancercomprising administering to an individual (e.g., a mammal, e.g., a humanor a nonhuman animal) in need thereof an effective amount of at leastone antibody, antibody fragment, or composition described herein.

In some embodiments, the antibody or antibody fragment binds to VISTA,thereby modulating or enhancing an immunogenic response to cancer. Insome embodiments, the cancer is a leukemia, lymphoma, myelodysplasticsyndrome and/or myeloma. In some embodiments, the cancer can be any kindor type of leukemia, including a lymphocytic leukemia or a myelogenousleukemia, such as, e.g., acute lymphoblastic leukemia (ALL), chroniclymphocytic leukemia (CLL), acute myeloid (myelogenous) leukemia (AML),chronic myelogenous leukemia (CML), hairy cell leukemia, T-cellprolymphocytic leukemia, large granular lymphocytic leukemia, or adultT-cell leukemia. In some embodiments, the lymphoma is a histocyticlymphoma, and in some embodiments, the cancer is a multiple myeloma. Insome embodiments, the cancer is a solid tumor, for example, a melanoma,or bladder cancer. In some embodiments, the cancer is a lung cancer(e.g., a non-small cell lung carcinoma (NSCLC)). Some methods oftreatment further comprise administering a vaccine (such as a viralvector vaccine, bacterial vaccine, cell-based vaccine, DNA vaccine, RNAvaccine, peptide vaccine, or protein vaccine). The invention alsorelates to a method for suppressing tumor growth in an individual inneed thereof, said method comprising administering an effective antibodyor antibody fragment or composition described herein.

The composition, antibody or fragment or other pharmaceutical agent(e.g., a vaccine) can be administered by any parenteral or nonparenteralmeans, for example, intravenously (IV), subcutaneously (SQ) or orally(PO).

In some embodiments, the composition, antibody or fragment isadministered quarterly, weekly, once every two weeks, once every threeweeks, or once every four weeks. In some embodiments, otherpharmaceutical or therapeutic agents are co-administered, before, duringor after the antibodies, fragments and compositions described herein.The co-administered agent can be administered by the same route as theantibody, fragment or composition, or by a different route.

The invention also includes methods of making the antibodies, fragmentsand compositions, for example, a method of producing an antibody orfragment described herein, comprising culturing host cells underconditions for production of said antibody. The method can furthercomprise isolating the antibody. The invention also relates to nucleicacids, e.g., isolated nucleic acids which comprises a nucleotidesequence encoding the antibodies and fragments, expression vectorscomprising such nucleic acids, e.g., operably linked to a promoter, andhost cells transformed with such an expression vector.

The invention also relates to kits and to articles of manufacturecomprising a composition comprising an anti-VISTA antibody and acontainer, and further comprising a package insert or label indicatingthat the composition can be used to treat cancer.

The invention also provides an isolated antibody or antibody fragmentthereof comprising an antigen binding region that binds to a V-domain IgSuppressor of T cell Activation (VISTA), wherein the antibody comprisesan antibody VH domain comprising a VH CDR1 having the amino acidsequence of SEQ ID NO:25, a VH CDR2 having the amino acid sequence ofSEQ ID NO:26 and a VH CDR3 having the amino acid sequence of SEQ IDNO:27, and which further comprises an antibody VL domain comprising a VLCDR1 having the amino acid sequence of SEQ ID NO:28, a VL CDR2 havingthe amino acid sequence of SEQ ID NO:29 and a VL CDR3 having the aminoacid sequence of SEQ ID NO:30. In some embodiments, the antibody orantibody fragment comprises one or more humanized or human frameworkregions. In particular embodiments, the antibody or antibody fragmentcomprises an antibody VH domain that comprises SEQ ID NO:37 and/or anantibody VL domain comprises SEQ ID NO:44. In certain embodiments, theantibody comprises a heavy chain constant region (e.g., a human heavychain constant region) and/or a light chain constant region (e.g., ahuman light chain constant region, such as the light chain constantregion present in SEQ ID NO:56). Preferably, the heavy chain constantregion is an IgG1 heavy chain constant region (e.g., the IgG1 heavychain constant region present in SEQ ID NO:61). Ina particularembodiment, the IgG1 heavy chain constant region has been modified toenhance protease resistance of the antibody. An example of an IgG1 heavychain constant region that has been modified to enhance proteaseresistance is the IgG1 heavy chain constant region present in SEQ IDNO:60. In certain embodiments, the antibody or antibody fragmentcomprises a heavy chain comprising SEQ ID NO:60 and a light chaincomprising SEQ ID NO:56; or a heavy chain comprising SEQ ID NO:61 and alight chain comprising SEQ ID NO:56. In a particular embodiment, theantibody or antibody fragment is expressed in a cell that is deficientin fucosylation enzymes (e.g., a Chinese hamster ovary (CHO) cell thatis deficient in fucosylation enzymes).

The invention also relates to a composition comprising an antibody orantibody fragment thereof comprising an antibody VH domain comprising aVH CDR1 having the amino acid sequence of SEQ ID NO:25, a VH CDR2 havingthe amino acid sequence of SEQ ID NO:26 and a VH CDR3 having the aminoacid sequence of SEQ ID NO:27, and which further comprises an antibodyVL domain comprising a VL CDR1 having the amino acid sequence of SEQ IDNO:28, a VL CDR2 having the amino acid sequence of SEQ ID NO:29 and a VLCDR3 having the amino acid sequence of SEQ ID NO:30; and apharmaceutically acceptable carrier, diluent, or excipient.

In another embodiment, the invention relates to a method for treatingcancer in an individual in need thereof, said method comprisingadministering to the subject an effective amount of the antibody orantibody fragment that binds to a V-domain Ig Suppressor of T cellActivation (VISTA), wherein the antibody comprises an antibody VH domaincomprising a VH CDR1 having the amino acid sequence of SEQ ID NO:25, aVH CDR2 having the amino acid sequence of SEQ ID NO:26 and a VH CDR3having the amino acid sequence of SEQ ID NO:27, and which furthercomprises an antibody VL domain comprising a VL CDR1 having the aminoacid sequence of SEQ ID NO:28, a VL CDR2 having the amino acid sequenceof SEQ ID NO:29 and a VL CDR3 having the amino acid sequence of SEQ IDNO:30. In a particular embodiment, the cancer is a lung cancer. In afurther embodiment, the lung cancer is a non-small cell lung carcinoma(NSCLC). In some embodiments, the method further comprises administeringa second cancer treatment (e.g., surgery, chemotherapy, radiationtherapy, biologic therapy, immunomodulatory therapy, and combinationsthereof).

The invention also provides an antibody or antibody fragment thereofcomprising an antigen binding region that binds to a V-domain IgSuppressor of T cell Activation (VISTA), wherein the antibody binds to aconformational epitope in VISTA (e.g., human VISTA). In someembodiments, the conformational epitope comprises, or is present within,residues 103-111 (NLTLLDSGL (SEQ ID N062)) and 136-146 (VQTGKDAPSNC (SEQID NO:63)) of human VISTA (SEQ ID NO:46). In another embodiment, theconformational epitope comprises, or is present within, residues 24-36(LLGPVDKGHDVTF (SEQ ID NO:64)), 54-65 (RRPIRDLTFQDL (SEQ ID NO:65) and100-102 (TMR) of human VISTA (SEQ ID NO:46). In yet another embodiment,the conformational epitope comprises amino acid residues in the FG loopof human VISTA (SEQ ID NO:46).

In addition, the invention relates to a method of enhancing an immuneresponse in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of an antibody thatbinds V-domain Ig Suppressor of T cell Activation (VISTA), or anantibody fragment thereof, comprising an antigen binding region thatbinds to VISTA, thereby enhancing an immune response to the cancer. In aparticular embodiment, the immune response is an antitumor immuneresponse.

In another embodiment, the invention provides a method of eliciting abiological response in an individual in need thereof, comprisingadministering to the individual a therapeutically effective amount of anantibody that binds V-domain Ig Suppressor of T cell Activation (VISTA),or an antibody fragment thereof, comprising an antigen binding regionthat binds to VISTA, thereby enhancing an immune response to the cancer.Examples of biological responses include activation of monocytes;induction of T-cell proliferation and cytokine secretion;antibody-dependent cell-mediated cytotoxicity (ADCC) of cells expressingVISTA; and antibody-dependent cellular phagocytosis (ADCP) of cellsexpressing VISTA.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A-1C: Graphs showing VISTA expression on TF1 AML Cells Expressionof VISTA protein by flow cytometry is shown in the TF-1 AML cell line.

FIG. 2A-2E: Graphs showing staining and gating strategies foridentification of Human Myeloid and Lymphoid Subsets.

FIG. 3A-3G: Graphs showing expression of VISTA on Human Myeloid andLymphoid Subsets from one healthy normal donor.

FIG. 4: Graph showing expression of VISTA on Human Myeloid and LymphoidSubsets across multiple healthy normal donors.

FIG. 5A-5B: Graph showing staining and gating strategies foridentification of expression of VISTA on Human Monocytes andMacrophages.

FIG. 6A-6C: Graphs showing expression of VISTA on Human Monocytes andMacrophages.

FIG. 7A-7E: Graphs showing staining and gating strategies foridentification of expression of VISTA on Human T and NK Cell Subsets.

FIG. 8A-8G: Graphs showing expression of VISTA on Human T and NKCellSubsets from one healthy normal donor.

FIG. 9: Graph showing expression of VISTA on Human T and NK Cell Subsetsacross multiple healthy normal donors.

FIG. 10A-10D: Graphs showing staining and gating strategies foridentification of expression of VISTA on Human Dendritic Cell subsets.

FIG. 11A-11C: Graphs showing expression of VISTA on Human Dendritic Cellsubsets and basophils from one healthy normal donor.

FIG. 12: Graph showing expression of VISTA on Human Dendritic CellSubsets and basophils across multiple healthy normal donors.

FIG. 13A-13D: Analysis of VISTA expression on healthy human peripheralblood cells. Profile of VISTA expression on healthy human peripheralblood cells using multicolor flow cytometry analysis: Whole bloodsamples from 2 different individuals were analyzed for VISTA expressionon (FIG. 13A) monocytesSSC^(lo)⋅CD11b^(hi)CD14^(hi)CD16^(−ve)CD33^(+ve)HLA-DR^(+ve)CD19^(−ve))(FIG. 13B) neutrophils (SSC^(hI)CD177⁺CD11b^(hi)CD14^(lo)CD16^(+ve)CD33^(+ve)HLA-DR^(−ve)CD19^(−ve)). Peripheral bloodmononuclear cells were isolated using Ficoll gradient for analysis of(FIG. 13C) CD4+ T cells (CD3^(+ve)CD4^(+ve)), and (FIG. 13D) CD8+ Tcells (CD3^(−ve)CD8^(+ve)).

FIG. 14A-14C: Analysis of VISTA expression on peripheral blood cellsfrom a lung cancer patient and a healthy control donor. Profile of VISTAexpression on lung cancer patient peripheral blood cells usingmulticolor flow cytometry analysis: Representative FACS plot (FIG. 14A)from one individual is shown. Peripheral blood mononuclear cells wereisolated by Ficoll and analyzed for VISTA expression on (FIG. 14B)monocytes (CD14+CD11b+CD33+HLADR+CD15−) and (FIG. 14C) myeloid derivedsuppressor cells (CD14−CD11b+CD33-HLADR-CD15+CD16+).

FIG. 15A-15C: Profile of VISTA expression in peripheral blood cells froma patient with colon cancer, using multicolor flow cytometry analysis:Representative FACS plot (FIG. 15A) from one individual is shown.Peripheral blood mononuclear cells were isolated by Ficoll and analyzedfor VISTA expression on (FIG. 15B) monocytes(CD14+CD11b+CD33+HLADR+CD15−) and (FIG. 15C) myeloid derived suppressorcells (CD14−CD11b+CD33−HLADR-CD15+CD16+).

FIG. 16A-16D: Profile of VISTA expression on Cynomolgus monkeyperipheral blood cells using multicolor flow cytometry analysis: Wholeblood from 4 different monkeys was analyzed for VISTA expression on(FIG. 16A) monocytes(SSC^(lo)CD11b^(hi)CD14^(hi)HLA-DR^(hi)CD16^(−ve)CD19^(−ve) and (FIG.16B) neutrophils CD11b^(hi)CD14^(lo) HLA-DR^(−ve)CD16^(−ve)CD19^(−ve).Peripheral blood mononuclear cells from three monkeys were isolatedusing Ficoll gradient for analysis of (FIG. 16C) CD4+ T cells(TCRa/(3^(+Ve)CD4⁺′) and (FIG. 16D) CD8+ T cells(TCRα/β^(+ve)CD8^(+ve)).

FIG. 17: Graph showing absolute expression values of VISTA RNA in Hemecell lines.

FIG. 18: Mouse A20 cells were stably transfected with either GFP orhuman VISTA. They were incubated with ova peptide and with DO11.10 Tcells. CD25 expression by the T cells was measured 24 hours afterincubation began. The A20-huVISTA cells suppress CD25 expression by theT cells, but this readout is significantly restored by incubation withVSTB95.

FIG. 19A-19F: Graphs showing Human VISTA ELISA results.

FIG. 20A-20F: Human VISTA FACS results, showing anti-VISTA antibodiesbinding to cells expressing human VISTA.

FIG. 21A-21D: Dilution study of 6 anti-VISTA antibody candidates in themixed lymphocyte reaction from 30 μg/ml to 0.0 μg/ml.

FIG. 22A-22B: Dilution studies of 6 anti-VISTA antibody candidates inthe SEB assay (individual CPM counts and IFN-g concentrations) from 30μg/ml to 0.0 μg/ml.

FIG. 23: Sensorgram plot using anti-VISTA antibody VSTB85 coated on aProteon SPR chip and VISTA protein with the indicated competitors runover the chip (competitors listed in Table 16).

FIG. 24: Experimental design for MB49 murine bladder tumor model

FIG. 25A-25B: MB49 tumor growth in female C57Bl/6 mice. Graphsillustrate tumor growth in individual mice treated with anti-mouse VISTAantibody (FIG. 25B) or control IgG (FIG. 25A).

FIG. 26: Amino acid sequence of human VISTA (SEQ ID NO:46).

FIG. 27: Multiple sequence alignment of VISTA orthologues

FIG. 28: Regions of human VISTA bound by VSTB50 and VSTB60 antibodies(top) or VSTB95 and VSTB112 antibodies (bottom), as determined by HDX

FIG. 29: VISTA Epitope bound by VSTB112. (Top) VISTA is shown in cartoonwith strands labeled. Residues having at least one atom within 5 Å ofVSTB112 in the complex are colored blue. Blue and orange sphereshighlight a chain break, and the cyan and green spheres mark the N- andC-termini of the VISTA structure, respectively. (Bottom) Sequence ofVISTA construct used in structure determination. Circles below thesequence are used to indicate residues which make only main chaincontacts with VSTB112, triangles indicate a side chain contact, andsquares indicate the side chain contact results in either a hydrogenbond or salt bridge interaction as calculated by PISA. Shapes arecolored to indicate the CDR having the greatest number of atomscontacted by the given residue with CDR colors defined in FIG. 59.Secondary structural elements are as defined in the program MOE withyellow arrows representing β-strands and red rectangles indicatingα-helices.

FIG. 30: VSTB112 Paratope. (Top) VISTA antigen is shown in illustrationand VSTB112 within 5 angstrom (Å) of VISTA is shown in surface withcolors used to designate CDR identity as specified in the sequencebelow. Contacting framework residues adjacent to a CDR are coloredsimilarly to the corresponding CDR (Bottom) Sequence of VSTB112 Fvregion. Colored backgrounds specify CDRs according to Kabat definitions.Circles below the sequence are used to indicate residues which make mainchain only contacts with VISTA, triangles indicate a side-chain contact,and squares indicate the side chain contact results in either a hydrogenbond or salt bridge interaction as calculated by PISA.

FIG. 31: Comparison of epitope regions identified by crystallography andhydrogen deuterium exchange (HDX). Sequence of VISTA construct used instructure determination. Circles below the sequence are used to indicateresidues which make only main chain contacts with VSTB112, trianglesindicate a side chain contact, and squares indicate the side chaincontact results in either a hydrogen bond or salt bridge interaction ascalculated by PISA.

FIG. 32: Activation of CD14+ monocytes in whole PBMC by VSTB174 (derivedfrom VSTB112). In each part of the experiment, cells were incubated withPBS, IgG1 control antibody, or VSTB174 at 1, 0.1 or 0.01 ug/ml. Leftpanel shows CD80 MFI; right panel shows HLA-DR MFI (two donors testedwith representative results shown).

FIG. 33: Graph showing ADCC activity of VSTB174 directed againstK562-VISTA cells.

FIG. 34: Graph showing ADCP activity of VSTB174 directed againstK562-VISTA cells. Both antibodies depicted have the same Fab, butVSTB174 has an IgG1 Fc and VSTB140 has Fc silent IgG2.

FIG. 35: Graph showing phagocytosis mediated by VSTB174, VSTB149 orVSTB140 mAbs against K562-VISTA. Each mAb was tested with 7 half logdoses, ranging from 0.0008 μg/ml to 0.56 ug/ml.

FIG. 36: Graph showing phagocytosis mediated by VSTB174, VSTB149 orVSTB140 mAbs against myeloma cell line K562 cells. Each mAb was testedwith 7 half log doses, ranging from 0.0008 μg/ml to 0.56 ug/ml.

FIG. 37: MB49 tumor efficacy study evaluating VSTB123 1, 5, 7.5, and 10mg/kg in female VISTA-KI mice. Tumor volumes were approximately 50 mm³when dosing began at day 6 after implant. VSTB123 is the VSTB112 Fabgrafted onto a mouse Fc scaffold and binds to human VISTA in theVISTA-KI mouse.

FIG. 38: Graph shows that CD14+ cells expressing high/intermediatelevels of VISTA are found in 13/13 lung cancer samples, as well as indistant lung tissue and peripheral blood of patients.

FIG. 39: IHC staining for VISTA in Lung Cancer using GG8.

DETAILED DESCRIPTION OF THE INVENTION

A description of example embodiments of the invention follows.

The present invention relates to antibodies to novel Immunoglobulinfamily ligand designated V-domain Immu{grave over (n)}oglobulinSuppressor of T cell Activation (VISTA) (Genbank: JN602184) (Wang etal., 2010, 2011). VISTA bears homology to PD-L1 but displays a uniqueexpression pattern that is restricted to the hematopoietic compartment.Specifically, VISTA is constitutively and highly expressed onCD11b^(high) myeloid cells, and expressed at lower levels on CD4⁺ andCD8⁺ T cells. The human homologue shares approximately 85% homology withmurine VISTA and has similar expression patterns (Lines et al., CancerResearch 74:1924, 2014). VISTA expressed on antigen presenting cells(APCs) suppresses CD4⁺ and CD8⁺ T cell proliferation and cytokineproduction via a cognate receptor independent of PD-1. In a passive EAE(experimental autoimmune encephalomyelitis) disease model, a VISTAspecific monoclonal antibody enhanced T-cell dependent immune responsesand exacerbated disease. VISTA over-expression on tumor cells impairedprotective anti-tumor immunity in tumor-bearing hosts. Studies of humanVISTA confirmed its suppressive function on human T cells (Lines et alCancer Research 74:1924, 2014. Studies from Flies et al. also identifiedVISTA (named PD-1H) as a potent immune suppressive molecule (Flies etal., 2011). VISTA is described in further detail in U.S. Publishedapplication US 20130177557 A1 and U.S. Pat. Nos. 7,919,585 and8,236,304, all of which are incorporated herein by reference in theirentirety.

VISTA is a novel negative checkpoint regulator that suppresses immuneresponses. As described in Example 12 herein, treatment with aVISTA-specific monoclonal antibody in murine tumor models has been shownto reverse the suppressive character of the tumor immunemicroenvironment and enhance protective anti-tumor immunity, thus,demonstrating the potential of a VISTA monoclonal antibody as a noveltherapeutic for cancer immunotherapy.

Antibodies and Fragments of the Present Invention

The term “antibody” is meant to include polyclonal antibodies,monoclonal antibodies (mAbs), chimeric antibodies, humanized antibodies,human antibodies and anti-idiotypic (anti-Id) antibodies, as well asfragments, regions or derivatives thereof, provided by any knowntechnique, such as, but not limited to, enzymatic cleavage, peptidesynthesis or recombinant techniques. Anti-VISTA antibodies of thepresent invention are capable of binding portions of VISTA thatmodulate, regulate, or enhance an immune response. In some embodiments,the antibodies competitively inhibit one or more of the anti-VISTAantibodies described herein. Methods for determining whether two or moreantibodies compete for binding to the same target are known in the art.For example, a competitive binding assay can be used to determinewhether one antibody blocks the binding of another antibody to thetarget. Typically, a competitive binding assay involves the use ofpurified target antigen (e.g., PD-1) bound either to a solid substrateor cells, an unlabeled test binding molecule, and a labeled referencebinding molecule. Competitive inhibition is measured by determining theamount of label bound to the solid surface or cells in the presence ofthe test binding molecule. Usually the test binding molecule is presentin excess. Typically, when a competing binding molecule is present inexcess, it will inhibit specific binding of a reference binding moleculeto a common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75%,or more. In some embodiments, competitive inhibition is determined usinga competitive inhibition ELISA assay.

Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of animals immunized with an antigen. Amonoclonal antibody contains a substantially homogeneous population ofantibodies specific to antigens, which population contains substantiallysimilar epitope binding sites. Monoclonal antibodies may be obtained bymethods known to those skilled in the art. See, for example Kohler andMilstein, Nature, 256:495-497 (1975); U.S. Pat. No. 4,376,110; Ausubelet al., eds., Current Protocols in Molecular Biology, Greene PublishingAssoc. and Wiley Interscience, N.Y., (1987, 1992); and Harlow and LaneANTIBODIES: A Laboratory Manual Cold Spring Harbor Laboratory (1988);Colligan et al., eds., Current Protocols in Immunology, GreenePublishing Assoc. and Wiley Interscience, N.Y., (1992, 1993), thecontents of all of which are incorporated entirely herein by reference.Such antibodies may be of any immunoglobulin class including IgG, IgM,IgE, IgA, GILD and any subclass thereof. A hybridoma producing amonoclonal antibody of the present invention may be cultivated in vitro,in situ or in vivo.

The invention also encompasses digestion fragments, specified portionsand variants thereof, including antibody mimetics or comprising portionsof antibodies that mimic the structure and/or function of an antibody orspecified fragment or portion thereof, including single chain antibodiesand fragments thereof. Functional fragments include antigen-bindingfragments that bind to a mammalian VISTA protein. For example, antibodyfragments capable of binding to VISTA or portions thereof, including,but not limited to Fab (e.g., by papain digestion), Fab′ (e.g., bypepsin digestion and partial reduction) and F(ab′)₂ (e.g., by pepsindigestion), facb (e.g., by plasmin digestion), pFc′ (e.g., by pepsin orplasmin digestion), Fd (e.g., by pepsin digestion, partial reduction andreaggregation), Fv or scFv (e.g., by molecular biology techniques)fragments, are encompassed by the invention (see, e.g., Colligan,Immunology, supra). Antibody fragments of the present invention alsoinclude those discussed and described in Aaron L. Nelson, mAbs 2:1,77-83 (January/February 2010), the contents of which are incorporated byreference in their entirety.

Such fragments can be produced, for example, by enzymatic cleavage,synthetic or recombinant techniques, as known in the art and/or asdescribed herein. antibodies can also be produced in a variety oftruncated forms using antibody genes in which one or more stop codonshave been introduced upstream of the natural stop site. For example, acombination gene encoding a F(ab′)₂ heavy chain portion can be designedto include DNA sequences encoding the CH1 domain and/or hinge region ofthe heavy chain. The various portions of antibodies can be joinedtogether chemically by conventional techniques, or can be prepared as acontiguous protein using genetic engineering techniques.

In one embodiment, the amino acid sequence of an immunoglobulin chain,or portion thereof (e.g., variable region, CDR) has about 70-100%identity (e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 orany range or value therein) to the amino acid sequence of thecorresponding variable sequence chain described herein. Preferably,70-100% amino acid identity (e.g., 85, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100 or any range or value therein) is determined using asuitable computer algorithm, as known in the art.

Examples of heavy chain and light chain variable regions sequences areprovided herein.

The antibodies of the present invention, or specified variants thereof,can comprise any number of contiguous amino acid residues from anantibody of the present invention, wherein that number is selected fromthe group of integers consisting of from 10-100% of the number ofcontiguous residues in an anti-TNF antibody. Optionally, thissubsequence of contiguous amino acids is at least about 10, 20, 30, 40,50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,200, 210, 220, 230, 240, 250 or more amino acids in length, or any rangeor value therein. Further, the number of such subsequences can be anyinteger selected from the group consisting of from 1 to 20, such as atleast 2, 3, 4, or 5.

As those of skill will appreciate, the present invention includes atleast one biologically active antibody of the present invention.Biologically active antibodies have a specific activity at least 20%,30%, or 40%, and preferably at least 50%, 60%, or 70%, and mostpreferably at least 80%, 90%, or 95%-100% of that of the native(non-synthetic), endogenous or related and known antibody. Methods ofassaying and quantifying measures of enzymatic activity and substratespecificity, are well known to those of skill in the art.

Substantial similarity refers to a compound having at least 85% (e.g.,at least 95%) identity and at least 85% (e.g., at least 95%) of activityof the native (non-synthetic), endogenous or related and known antibody.

As used herein, the term “human antibody” refers to an antibody in whichsubstantially every part of the protein (e.g., CDR, framework, CL, CHdomains (e.g., CH1, CH2, CH3), hinge, (VL, VH)) is substantiallynon-immunogenic in humans, with only minor sequence changes orvariations. Similarly, antibodies designated primate (monkey, baboon,chimpanzee, and the like), rodent (mouse, rat, and the like) and othermammals designate such species, sub-genus, genus, sub-family, familyspecific antibodies. Further, chimeric antibodies can include anycombination of the above. Such changes or variations optionally andpreferably retain or reduce the immunogenicity in humans or otherspecies relative to non-modified antibodies. Thus, a human antibody isdistinct from a chimeric or humanized antibody. It is pointed out that ahuman antibody can be produced by a non-human animal or prokaryotic oreukaryotic cell that is capable of expressing functionally rearrangedhuman immunoglobulin (e.g., heavy chain and/or light chain) genes.Further, when a human antibody is a single chain antibody, it cancomprise a linker peptide that is not found in native human antibodies.For example, an Fv can comprise a linker peptide, such as two to abouteight glycine or other amino acid residues, which connects the variableregion of the heavy chain and the variable region of the light chain.Such linker peptides are considered to be of human origin.

Bispecific, heterospecific, heteroconjugate or similar antibodies canalso be used that are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is forat least one VISTA protein, the other one is for any other antigen.Methods for making bispecific antibodies are known in the art. Therecombinant production of bispecific antibodies can be based on theco-expression of two immunoglobulin heavy chain-light chain pairs, wherethe two heavy chains have different specificities (Milstein and Cuello,Nature 305:537 (1983)). See also WO 93/08829, U.S. Pat. Nos. 6,210,668,6,193,967, 6,132,992, 6,106,833, 6,060,285, 6,037,453, 6,010,902,5,989,530, 5,959,084, 5,959,083, 5,932,448, 5,833,985, 5,821,333,5,807,706, 5,643,759, 5,601,819, 5,582,996, 5,496,549, 4,676,980, WO91/00360, WO 92/00373, EP 03089, Traunecker et al., EMBO J. 10:3655(1991), Suresh et al., Methods in Enzymology 121:210 (1986), eachentirely incorporated herein by reference.

In one embodiment, the invention relates to a bispecific antibodytargeting VISTA and a second target protein (e.g., an immune checkpointprotein). Exemplary bispecific anitbodies include a bispecific antibodytargeting VISTA and PD-L1 and a bispecific antibody targeting VISTA andPD-L2.

Human antibodies that are specific for human VISTA proteins or fragmentsthereof can be raised against an appropriate immunogenic antigen, suchas VISTA protein or a portion thereof (including synthetic molecules,such as synthetic peptides).

Other specific or general mammalian antibodies can be similarly raised.Immunogenic antigens preparation and monoclonal antibody production canbe performed using any suitable technique.

For example, a hybridoma is produced by fusing a suitable immortal cellline (e.g., a myeloma cell line such as, but not limited to, Sp2/0,Sp2/0-AG14, NSO, NS1, NS2, AE-1, L.5, >243, P3X63Ag8.653, Sp2 SA3, Sp2MAI, Sp2 SS1, Sp2 SA5, U937, MLA 144, ACT IV, MOLT4, DA-1, JURKAT, WEHI,K-562, COS, RAJI, NIH 3T3, HL-60, MLA 144, NAMAIWA, NEURO 2A, or thelike, or heteromylomas, fusion products thereof, or any cell or fusioncell derived therefrom, or any other suitable cell line as known in theart, See, e.g., www.atcc.org, with antibody-producing cells.Antibody-producing cells can include isolated or cloned spleen,peripheral blood, lymph, tonsil, or other immune cells (e.g., B cells),or any other cells expressing heavy or light chain constant or variableor framework or complementarity determining region (CDR) sequences. Suchantibody-producing cells can be recombinant or endogenous cells, and canalso be prokaryotic or eukaryotic (e.g., mammalian, such as, rodent,equine, ovine, goat, sheep, primate). See, e.g., Ausubel, supra, andColligan, Immunology, supra, chapter 2, entirely incorporated herein byreference.

Antibody producing cells can also be obtained from the peripheral bloodor, preferably the spleen or lymph nodes, of humans or other suitableanimals that have been immunized with the antigen of interest. Any othersuitable host cell can also be used for expressing heterologous orendogenous nucleic acid encoding an antibody, specified fragment orvariant thereof, of the present invention. Fused cells (hybridomas) orrecombinant cells can be isolated using selective culture conditions orother suitable known methods, and cloned by limiting dilution or cellsorting, or other known methods. Cells which produce antibodies with thedesired specificity can be selected by a suitable assay (e.g.,enzyme-linked immunosorbent assay (ELISA)).

Other suitable methods of producing or isolating antibodies of therequisite specificity can be used, including, but not limited to,methods that select recombinant antibody from a peptide or proteinlibrary (e.g., but not limited to, a bacteriophage, ribosome,oligonucleotide, RNA, cDNA, or the like, display library; e.g., asavailable from Cambridge antibody Technologies, Cambridgeshire, UK;MorphoSys, Martinsreid/Planegg, DE; Biovation; Aberdeen, Scotland, UK;Bioinvent, Lund, Sweden; Dyax Corp., Enzon, Affymax/Biosite; Xoma,Berkeley, Calif.; Ixsys. See, e.g., PCT/GB91/01134; PCT/GB92/01755;PCT/GB92/002240; PCT/GB92/00883; PCT/GB93/00605; PCT/GB94/01422;PCT/GB94/02662; PCT/GB97/01835; WO90/14443; WO90/14424; WO90/14430;PCT/US94/1234; WO92/18619; WO96/07754; EP 614 989; WO95/16027;WO88/06630; WO90/3809; U.S. Pat. No. 4,704,692; PCT/US91/02989;WO89/06283; EP 371 998; EP 550 400; EP 229 046; PCT/US91/07149; orstochastically-generated peptides or proteins—U.S. Pat. Nos. 5,723,323;5,763,192; 5,814,476; 5,817,483; 5,824,514; 5,976,862; WO 86/05803, EP590 689, each entirely incorporated herein by reference, or that relyupon immunization of transgenic animals (e.g., SCID mice, Nguyen et al.,Microbiol. Immunol. 41:901-907 (1997); Sandhu et al., Crit. Rev.Biotechnol. 16:95-118 (1996); Eren et al., Immunol. 93:154-161 (1998),each entirely incorporated by reference as well as related patents andapplications) that are capable of producing a repertoire of humanantibodies, as known in the art and/or as described herein. Suchtechniques, include, but are not limited to, ribosome display (Hanes etal., Proc. Natl. Acad. Sci. USA, 94:4937-4942 (May 1997); Hanes et al.,Proc. Natl. Acad. Sci. USA, 95:14130-14135 (November 1998)); single cellantibody producing technologies (U.S. Pat. No. 5,627,052, Wen et al., J.Immunol. 17:887-892 (1987); Babcook et al., Proc. Natl. Acad. Sci. USA93:7843-7848 (1996)); gel microdroplet and flow cytometry (Powell etal., Biotechnol. 8:333-337 (1990); One Cell Systems, Cambridge, Mass.;Gray et al., J. Imm. Meth. 182:155-163 (1995); Kenny et al.,Bio/Technol. 13:787-790 (1995)); B-cell selection (Steenbakkers et al.,Molec. Biol. Reports 19:125-134 (1994); Jonak et al., Progress Biotech,Vol. 5, In Vitro Immunization in Hybridoma Technology, Borrebaeck, ed.,Elsevier Science Publishers B.V., Amsterdam, Netherlands (1988)).

Methods for engineering or humanizing non-human or human antibodies canalso be used and are well known in the art. Generally, a humanized orengineered antibody has one or more amino acid residues from a sourcewhich is non-human, e.g., but not limited to mouse, rat, rabbit,non-human primate or other mammal. These human amino acid residues areoften referred to as “import” residues, which are typically taken froman “import” variable, constant or other domain of a known humansequence. Known human Ig sequences are disclosed, e.g.,www.ncbi.nlm.nih.gov/entrez/query.fcgi; www.atcc.org/phage/hdb.html,each entirely incorporated herein by reference.

Such imported sequences can be used to reduce immunogenicity or reduce,enhance or modify binding, affinity, avidity, specificity, half-life, orany other suitable characteristic, as known in the art. Generally partor all of the non-human or human CDR sequences are maintained while partor all of the non-human sequences of the framework and/or constantregions are replaced with human or other amino acids. Antibodies canalso optionally be humanized with retention of high affinity for theantigen and other favorable biological properties usingthree-dimensional immunoglobulin models that are known to those skilledin the art. Computer programs are available which illustrate and displayprobable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of the residues in the functioning of thecandidate immunoglobulin sequence, i.e., the analysis of residues thatinfluence the ability of the candidate immunoglobulin to bind itsantigen. In this way, framework (FR) residues can be selected andcombined from the consensus and import sequences so that the desiredantibody characteristic, such as increased affinity for the targetantigen(s), is achieved. In general, the CDR residues are directly andmost substantially involved in influencing antigen binding. Humanizationor engineering of antibodies of the present invention can be performedusing any known method, such as but not limited to those described in,for example, Winter (Jones et al., Nature 321:522 (1986); Riechmann etal., Nature 332:323 (1988); Verhoeyen et al., Science 239:1534 (1988)),Sims et at, J. Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol.Biol. 196:901 (1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A.89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993), U.S. Pat.Nos. 5,723,323, 5,976,862, 5,824514, 5,817483, 5,814476, 5,763,192,5,723,323, 5,766,886, 5,714,352, 6,204,023, 6,180,370, 5,693,762,5,530,101, 5,585,089, 5,225,539; 4,816,567, each entirely incorporatedherein by reference, included references cited therein.

The anti-VISTA antibody can also be optionally generated by immunizationof a transgenic animal (e.g., mouse, rat, rabbit, hamster, non-humanprimate, and the like) capable of producing a repertoire of humanantibodies, as described herein and/or as known in the art. Cells thatproduce a human anti-VISTA antibody can be isolated from such animalsand immortalized using suitable methods, such as the methods describedherein.

Transgenic animals that can produce a repertoire of human antibodiesthat bind to human antigens can be produced by known methods (e.g., butnot limited to, U.S. Pat. Nos. 5,770,428, 5,569,825, 5,545,806,5,625,126, 5,625,825, 5,633,425, 5,661,016 and 5,789,650 issued toLonberg et al.; Jakobovits et al. WO 98/50433, Jakobovits et al. WO98/24893, Lonberg et al. WO 98/24884, Lonberg et al. WO 97/13852,Lonberg et al. WO 94/25585, Kucherlapate et al. WO 96/34096,Kucherlapate et al. EP 0463 151 B1, Kucherlapate et al. EP 0710 719 A1,Surani et al. U.S. Pat. No. 5,545,807, Bruggemann et al. WO 90/04036,Bruggemann et al. EP 0438 474 B1, Lonberg et al. EP 0814 259 A2, Lonberget al. GB 2 272 440 A, Lonberg et al. Nature 368:856-859 (1994), Tayloret al., Int. Immunol. 6(4)579-591 (1994), Green et al, Nature Genetics7:13-21 (1994), Mendez et al., Nature Genetics 15:146-156 (1997), Tayloret al., Nucleic Acids Research 20(23):6287-6295 (1992), Tuaillon et al.,Proc Natl Acad Sci USA 90(8)3720-3724 (1993), Lonberg et al., Int RevImmunol 13(1):65-93 (1995) and Fishwald et al., Nat Biotechnol14(7):845-851 (1996), which are each entirely incorporated herein byreference). Generally, these mice comprise at least one transgenecomprising DNA from at least one human immunoglobulin locus that isfunctionally rearranged, or which can undergo functional rearrangement.The endogenous immunoglobulin loci in such mice can be disrupted ordeleted to eliminate the capacity of the animal to produce antibodiesencoded by endogenous genes.

Screening antibodies for specific binding to similar proteins orfragments can be conveniently achieved using peptide display libraries.This method involves the screening of large collections of peptides forindividual members having the desired function or structure. Antibodyscreening of peptide display libraries is well known in the art. Thedisplayed peptide sequences can be from 3 to 5000 or more amino acids inlength, frequently from 5-100 amino acids long, and often from about 8to 25 amino acids long. In addition to direct chemical synthetic methodsfor generating peptide libraries, several recombinant DNA methods havebeen described. One type involves the display of a peptide sequence onthe surface of a bacteriophage or cell. Each bacteriophage or cellcontains the nucleotide sequence encoding the particular displayedpeptide sequence. Such methods are described in PCT Patent PublicationNos. 91/17271, 91/18980, 91/19818, and 93/08278. Other systems forgenerating libraries of peptides have aspects of both in vitro chemicalsynthesis and recombinant methods. See, PCT Patent Publication Nos.92/05258, 92/14843, and 96/19256. See also, U.S. Pat. Nos. 5,658,754;and 5,643,768. Peptide display libraries, vector, and screening kits arecommercially available from such suppliers as Invitrogen (Carlsbad,Calif.), and Cambridge antibody Technologies (Cambridgeshire, UK). See,e.g., U.S. Pat. Nos. 4,704,692, 4,939,666, 4,946,778, 5,260,203,5,455,030, 5,518,889, 5,534,621, 5,656,730, 5,763,733, 5,767,260,5,856,456; 5,223,409, 5,403,484, 5,571,698, 5,837,500, assigned to Dyax,U.S. Pat. Nos. 5,427,908, 5,580,717; 5,885,793, assigned to Cambridgeantibody Technologies; U.S. Pat. No. 5,750,373, assigned to Genentech,U.S. Pat. Nos. 5,618,920, 5,595,898, 5,576,195, 5,698,435, 5,693,493,and 5,698,417.

Antibodies of the present invention can also be prepared using at leastone anti-VISTA antibody encoding nucleic acid to provide transgenicanimals, such as goats, cows, sheep, and the like, that produce suchantibodies in their milk. Such animals can be provided using knownmethods. See, e.g., but not limited to, U.S. Pat. Nos. 5,827,690;5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362; 5,304,489, andthe like, each of which is entirely incorporated herein by reference.

The anti-VISTA antibodies of the present invention can also be producedusing transgenic plants, according to known methods. See also, e.g.,Fischer et al., Biotechnol. Appl. Biochem. 30:99-108 (October, 1999),Cramer et al., Curr. Top. Microbol. Immunol. 240:95-118 (1999) andreferences cited therein; Ma et al., Trends Biotechnol. 13:522-7 (1995);Ma et al., Plant Physiol. 109:341-6 (1995); Whitelam et al., Biochem.Soc. Trans. 22:940-944 (1994); and references cited therein. Each of theabove references is entirely incorporated herein by reference.

The antibodies of the invention can bind human VISTA with a wide rangeof affinities (K_(D)). In a preferred embodiment, at least one humanmonoclonal antibody of the present invention can optionally bind humanVISTA with high affinity. For example, a human monoclonal antibody canbind human VISTA with a K_(D) equal to or less than about 10⁻⁷ M, suchas but not limited to, 0.1-9.9 (or any range or value therein)×10⁻⁷,10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, 10⁻¹², 10⁻¹³ or any range or value therein. Insome embodiments, the antibody or antibody fragment can binds humanVISTA with an affinity of at least 1×10⁻⁷ liter/mole, for example, atleast 1×10⁻⁸ liter/mole, for example, at least 1×10⁻⁹ liter/moleliter/mole.

The affinity or avidity of an antibody for an antigen can be determinedexperimentally using any suitable method. (See, for example, Berzofsky,et al., “Antibody-Antigen Interactions,” In Fundamental Immunology,Paul, W. E., Ed., Raven Press: New York, N.Y. (1984); Kuby, JanisImmunology, W.H. Freeman and Company: New York, N.Y. (1992); and methodsdescribed herein). The measured affinity of a particularantibody-antigen interaction can vary if measured under differentconditions (e.g., salt concentration, pH). Thus, measurements ofaffinity and other antigen-binding parameters (e.g., K_(D), K_(a),K_(d)) are preferably made with standardized solutions of antibody andantigen, and a standardized buffer.

Nucleic Acid Molecules

Using the information provided herein, such as the nucleotide sequencesencoding at least 70-100% of the contiguous amino acids of at least oneof specified fragments, variants or consensus sequences thereof, or adeposited vector comprising at least one of these sequences, a nucleicacid molecule of the present invention encoding at least one anti-VISTAantibody comprising all of the heavy chain variable CDR regions of SEQID NOS:1, 2 and 3 and/or all of the light chain variable CDR regions ofSEQ ID NOS:4, 5 and 6 can be obtained using methods described herein oras known in the art.

Nucleic acid molecules of the present invention can be in the form ofRNA, such as mRNA, hnRNA, tRNA or any other form, or in the form of DNA,including, but not limited to, cDNA and genomic DNA obtained by cloningor produced synthetically, or any combinations thereof. The DNA can betriple-stranded, double-stranded or single-stranded, or any combinationthereof. Any portion of at least one strand of the DNA or RNA can be thecoding strand, also known as the sense strand, or it can be thenon-coding strand, also referred to as the anti-sense strand.

Isolated nucleic acid molecules of the present invention can includenucleic acid molecules comprising an open reading frame (ORF), forexample, but not limited to, at least one specified portion of at leastone CDR, as CDR1, CDR2 and/or CDR3 of at least one heavy chain or lightchain; nucleic acid molecules comprising the coding sequence for ananti-VISTA antibody or fragment, e.g., a fragment comprising a variableregion; and nucleic acid molecules which comprise a nucleotide sequencedifferent from those described above but which, due to the degeneracy ofthe genetic code, still encode at least one anti-VISTA antibody asdescribed herein and/or as known in the art. It would be routine for oneskilled in the art to generate such degenerate nucleic acid variantsthat code for specific anti-VISTA antibodies of the present invention.See, e.g., Ausubel, et al., supra, and such nucleic acid variants areincluded in the present invention.

As indicated herein, nucleic acid molecules of the present inventionwhich comprise a nucleic acid encoding an anti-VISTA antibody caninclude, but are not limited to, those encoding the amino acid sequenceof an antibody fragment; the coding sequence for the entire antibody ora portion thereof; the coding sequence for an antibody, fragment orportion, as well as additional sequences, such as the coding sequence ofat least one signal leader or fusion peptide, with or without theaforementioned additional coding sequences, such as at least one intron,together with additional, non-coding sequences, including but notlimited to, non-coding 5′ and 3′ sequences, such as the transcribed,non-translated sequences that play a role in transcription, mRNAprocessing, including splicing and polyadenylation signals (forexample—ribosome binding and stability of mRNA); an additional codingsequence that codes for additional amino acids, such as those thatprovide additional functionalities. Thus, the sequence encoding anantibody can be fused to a marker sequence, such as a sequence encodinga peptide that facilitates purification of the fused antibody comprisingan antibody fragment or portion.

Human genes which encode the constant (C) regions of the antibodies,fragments and regions of the present invention can be derived from ahuman fetal liver library, by known methods. Human C regions genes canbe derived from any human cell including those which express and producehuman immunoglobulins. The human C_(H) region can be derived from any ofthe known classes or isotypes of human H chains, including γ, μ, α, δ orε and subtypes thereof, such as G1, G2, G3 and G4. Since the H chainisotype is responsible for the various effector functions of anantibody, the choice of C_(H) region will be guided by the desiredeffector functions, such as complement fixation, or activity inantibody-dependent cellular cytotoxicity (ADCC).

Compositions

The pharmaceutical compositions disclosed herein are prepared inaccordance with standard procedures and are administered at dosages thatare selected to treat, e.g., reduce, prevent, or eliminate, or to slowor halt the progression of, the condition being treated (See, e.g.,Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton,Pa., and Goodman and Gilman's The Pharmaceutical Basis of Therapeutics,McGraw-Hill, New York, N.Y., the contents of which are incorporatedherein by reference, for a general description of the methods foradministering various agents for human therapy). The compositionscomprising the disclosed antibodies and agents can be delivered usingcontrolled or sustained-release delivery systems (e.g., capsules,biodegradable matrices). Examples of delayed-release delivery systemsfor drug delivery that would be suitable for administration of thecompositions of the disclosed compounds are described in, e.g., U.S.Pat. Nos. U.S. Pat. Nos. 5,990,092; 5,039,660; 4,452,775; and 3,854,480,the entire teachings of which are incorporated herein by reference.

For preparing pharmaceutical compositions from the anti-VISTA antibodiesand/or fragments of the present invention, pharmaceutically acceptablecarriers can be solid or liquid. Solid form preparations includepowders, tablets, pills, capsules, cachets, suppositories, anddispersible granules. For example, the compounds of the presentinvention can be in powder form for reconstitution at the time ofdelivery. A solid carrier can be one or more substances which can alsoact as diluents, flavoring agents, solubilizers, lubricants, suspendingagents, binders, preservatives, tablet disintegrating agents, or anencapsulating material. In powders, the carrier is a finely dividedsolid which is in a mixture with the finely divided active ingredient.

The powders and tablets preferably contain from about one to aboutseventy percent of the active ingredient. Suitable carriers aremagnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin,dextrin, starch, gelatin, tragacanth, methylcellulose, sodiumcaboxymethylcellulose, a low-melting wax, cocoa butter, and the like.Tablets, powders, cachets, lozenges, fast-melt strips, capsules andpills can be used as solid dosage forms containing the active ingredientsuitable for oral administration.

Liquid form preparations include solutions, suspensions, retentionenemas, and emulsions, for example, water or water propylene glycolsolutions. For parenteral injection, liquid preparations can beformulated in solution in aqueous polyethylene glycol solution.

The pharmaceutical composition can be in unit dosage form. In such form,the composition is subdivided into unit doses containing appropriatequantities of the active ingredient. The unit dosage form can be apackaged preparation, the package containing discrete quantities of unitdoses. The dosages can be varied depending upon the requirements of thepatient, the severity of the condition being treated, the compound andthe route of administration being employed. Determination of the properdosage for a particular situation is within the skill in the art.

Also, the pharmaceutical composition can contain, if desired, othercompatible agents, e.g., pharmaceutical, therapeutic or prophylacticagents. Therapeutic or prophylactic agents include, but are not limitedto, peptides, polypeptides, proteins, fusion proteins, nucleic acidmolecules, small molecules, mimetic agents, synthetic drugs, inorganicmolecules, and organic molecules. Examples of the classes of such agents(e.g., anti-cancer agents) include, but are not limited to, cytotoxins,angiogenesis inhibitors, immunomodulatory agents, immuno-oncologyagents, and agents used to provide relief from pain or to offset thedeleterious effects of one or more therapeutic agents (e.g.,bisphosphonate use to reduce the hypercalcemic effects ofglucocorticoids).

Angiogenesis inhibitors, agents and therapies that are suitable for usein the compositions and methods described herein include, but are notlimited to, angiostatin (plasminogen fragment); antiangiogenicantithrombin III; angiozyme. Bisphosphonates include, but are notlimited to, alendronate, clodronate, etidronate, ibandronate,pamidronate, risedronate, tiludronate, and zoledronate.

Immunomodulatory agents and therapies that are suitable for use in thecompositions and methods described herein include, but are not limitedto, anti-T cell receptor antibodies such as anti-CD3 antibodies (e.g.Nuvion (Protein Design Labs), OKT3 (Johnson & Johnson), or anti-CD20antibodies Rittman (IDEC)), anti-CD52 antibodies (e.g. CAMPATH 1H(Ilex)), anti-CD11a antibodies (e.g. Xanelim (Genentech)); anti-cytokineor anti-cytokine receptor antibodies and antagonists such as anti-IL-2receptor antibodies (Zenapax (Protein Design Labs)), anti-IL-6 receptorantibodies (e.g. MRA (Chugai)), and anti-IL-12 antibodies(CNT01275(Janssen)), anti-TNFalpha antibodies (Remicade(Janssen)) or TNFreceptor antagonist (Enbrel (Immunex)), anti-IL-6 antibodies (BE8(Diaclone) and siltuximab (CNTO32 (Centocor)), and antibodies thatimmunospecifically bind to tumor-associated antigens (e.g., trastuzimab(Genentech)).

Immuno-oncology agents that are suitable for use in the compositions andmethods described herein include, but are not limited to, ipilimumab(anti-CTLA-4), nivolumab (anti-PD-1), pembrolizumab (anti-PD-1),anti-PD-L1 antibodies, and anti-LAG-3 antibodies.

The composition is preferably made in the form of a dosage unitcontaining a therapeutically effective amount of the antibody orfragment. Examples of dosage units are tablets and capsules. Fortherapeutic purposes, the tablets and capsules can contain, in additionto the active ingredient, conventional carriers such as binding agents,for example, acacia gum, gelatin, polyvinylpyrrolidone, sorbitol, ortragacanth; fillers, for example, calcium phosphate, glycine, lactose,maize-starch, sorbitol, or sucrose; lubricants, for example, magnesiumstearate, polyethylene glycol, silica, or talc; disintegrants, forexample potato starch, flavoring or coloring agents, or acceptablewetting agents. Oral liquid preparations generally in the form ofaqueous or oily solutions, suspensions, emulsions, syrups or elixirs cancontain conventional additives such as suspending agents, emulsifyingagents, non-aqueous agents, preservatives, coloring agents and flavoringagents. Examples of additives for liquid preparations include acacia,almond oil, ethyl alcohol, fractionated coconut oil, gelatin, glucosesyrup, glycerin, hydrogenated edible fats, lecithin, methyl cellulose,methyl or propyl para-hydroxybenzoate, propylene glycol, sorbitol, orsorbic acid.

Other general details regarding methods of making and using thecompounds and compositions described herein are well-known in the art.See, e.g., U.S. Pat. No. 7,820,169, the contents of which areincorporated in their entirely.

Methods of Treatment

One of skill in the art, e.g., a clinician, can determine the suitabledosage and route of administration for a particular antibody, fragmentor composition for administration to an individual, considering theagents chosen, pharmaceutical formulation and route of administration,various patient factors and other considerations. Preferably, the dosagedoes not cause or produces minimal or no adverse side effects. Instandard multi-dosing regimens, a pharmacological agent may beadministered on a dosage schedule that is designed to maintain apre-determined or optimal plasma concentration in the subject undergoingtreatment. The antibodies, fragments and compositions can be added atany appropriate dosage ranges or therapeutically effective amount, forexample, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2.0 mg/kg,2.5 mg/kg, 3.0 mg/kg, 4.0 mg/kg, 5.0 mg/kg, 6.0 mg/kg, 7.0 mg/kg, 8.0mg/kg, 9.0 mg/kg, 10.0 mg/kg, 11.0 mg/kg, 12.0 mg/kg, 13.0 mg/kg, 14.0mg/kg, 15.0 mg/kg, 16.0 mg/kg, 17.0 mg/kg, 18.0 mg/kg, 19.0 mg/kg, 20.0mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg 60 mg/kg, 70 mg/kg, 80 mg/kg, 90mg/kg and 100 mg/kg. In one embodiment, the dosage of the administeredcomposition, antibody or fragment is 0.1-15 mg/kg per administration.

The antibody or fragment can be administered once, at least once, twice,at least twice, three times, or at least three times per day. Theantibody or fragment can be administered once, at least once, twice, atleast twice, three times, at least three times, four times, at leastfour times, five times, at least five times, six times per week, or atleast six times per week. The antibody or fragment can be administeredonce per month, at least once per month, twice per month, at least twiceper month, three times per month or at least three times per month. Theantibody or antibody fragment can be administered once per year, atleast once per year, twice per year, at least twice per year, threetimes per year, at least three times per year, four times per year, atleast four times per year, five times per year, at least five times peryear, six times per year or at least six times per year.

The anti-VISTA antibodies, fragments and compositions can, for example,be administered through parenteral or nonparenteral means, including,but not limited to, intravenously, subcutaneously, orally, rectally,intramuscularly, intraperitoneally, transmucosally, transdermally,intrathecally, nasally, or topically. One of ordinary skill in the artwill recognize that the following dosage forms can comprise as theactive ingredient, either compounds or a corresponding pharmaceuticallyacceptable salt of a compound of the present invention. In someembodiments, the dosage forms can comprise as the active ingredient,either a compound or a corresponding pharmaceutically acceptable salt ofa compound.

The anti-VISTA antibodies of the invention can be administered as partof a combination therapy (e.g., with each other, or with one or moreother therapeutic agents). The compounds of the invention can beadministered before, after or concurrently with one or more othertherapeutic agents. In some embodiments, a compound of the invention andother therapeutic agent can be co-administered simultaneously (e.g.,concurrently) as either separate formulations or as a joint formulation.Alternatively, the agents can be administered sequentially, as separatecompositions, within an appropriate time frame, as determined by theskilled clinician (e.g., a time sufficient to allow an overlap of thepharmaceutical effects of the therapies). A compound of the inventionand one or more other therapeutic agents can be administered in a singledose or in multiple doses, in an order and on a schedule suitable toachieve a desired therapeutic effect.

The present invention also provides a method for modulating or treatingat least one malignant disease in a cell, tissue, organ, animal orpatient. In some embodiments, the compounds and compositions of thepresent invention are used to treat or prevent cancer. Cancer caninclude any malignant or benign tumor of any organ or body system.Examples include, but are not limited to, the following: breast,digestive/gastrointestinal, endocrine, neuroendocrine, eye,genitourinary, germ cell, gynecologic, head and neck, hematologic/blood,musculoskeletal, neurologic, respiratory/thoracic, bladder, colon,rectal, lung, endometrial, kidney, pancreatic, liver, stomach,testicular, esophageal, prostate, brain, cervical, ovarian and thyroidcancers. Other, cancers can include leukemias, melanomas, and lymphomas,and any cancer described herein. In some embodiments, the solid tumor isinfiltrated with myeloid and/or T-cells. In some embodiments, the canceris a leukemia, lymphoma, myelodysplastic syndrome and/or myeloma. Insome embodiments, the cancer can be any kind or type of leukemia,including a lymphocytic leukemia or a myelogenous leukemia, such as,e.g., acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia(CLL), acute myeloid (myelogenous) leukemia (AML), chronic myelogenousleukemia (CML), hairy cell leukemia, T-cell prolymphocytic leukemia,large granular lymphocytic leukemia, or adult T-cell leukemia. In someembodiments, the lymphoma is a histocytic lymphoma, follicular lymphomaor Hodgkin lymphoma, and in some embodiments, the cancer is a multiplemyeloma. In some embodiments, the cancer is a solid tumor, for example,a melanoma, or bladder cancer. In a particular embodiment, the cancer isa lung cancer, such as a non-small cell lung cancer (NSCLC).

The present invention also provides a method for modulating or treatingat least one malignant disease in a cell, tissue, organ, animal orpatient, including, but not limited to, at least one of: leukemia, acuteleukemia, acute lymphoblastic leukemia (ALL), B-cell, T-cell or FAB ALL,acute myeloid leukemia (AML), chronic myelocytic leukemia (CML), chroniclymphocytic leukemia (CLL), hairy cell leukemia, myelodysplasticsyndrome (MDS), a lymphoma, Hodgkin's disease, a malignant lymphoma,non-hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, Kaposi'ssarcoma, colorectal carcinoma, pancreatic carcinoma, nasopharyngealcarcinoma, malignant histiocytosis, paraneoplasticsyndrome/hypercalcemia of malignancy, solid tumors, adenocarcinomas,sarcomas, malignant melanoma, hemangioma, metastatic disease, cancerrelated bone resorption, cancer-related bone pain, and the like. In someembodiments, the solid tumor is infiltrated with myeloid and/or T-cells.In a particular embodiment, the solid tumor is a lung cancer, such as anon-small cell lung cancer (NSCLC).

In some embodiments, the compounds and therapies described herein areco-administered with a vaccine (such as a viral vector vaccine,bacterial vaccine, cell-based vaccine, DNA vaccine, RNA vaccine, peptidevaccine, or protein vaccine). Such vaccines are well known in the art.See, e.g., Jeffrey Schlom, “Therapeutic Cancer Vaccines: Current Statusand Moving Forward,” J Natl Cancer Inst; 104:599-613 (2012), thecontents of which are incorporated herein in their entirely.

In some embodiments, the compounds and therapies described herein areco-administered with agents for chemotherapy, hormone therapies andbiological therapies, and/or bisphosphonates. In some embodiments, theagent(s) for chemotherapy include one or more of the following:arboplatin (Paraplatin), cisplatin (Platinol, Platinol-AQ),cyclophosphamide (Cytoxan, Neosar), doxorubicin (Adriamycin), etoposide(VePesid), fluorouracil (5-FU), gemcitabine (Gemzar), irinotecan(Camptosar), paclitaxel (Taxol), topotecan (Hycamtin), vincristine(Oncovin, Vincasar PFS), vinblastine (Velban).

In other embodiments, the anti-VISTA compounds and therapies describedherein are co-administered with one or more immune checkpointantibodies, such as, for example, nivolumab, pembrolizumab,tremelimumab, ipilimumab, anti-PD-L1 antibody, anti-PD-L2 antibody,anti-TIM-3 antibody, anti-LAG-3v, anti-OX40 antibody and anti-GITRantibody.

In another embodiment, the anti-VISTA compounds and therapies describedherein are co-administered with a small molecule inhibitor ofindoleamine 2,3-dioxygenase (IDO).

The anti-VISTA compounds and composition of the invention may beadministered to a subject in need thereof to prevent (includingpreventing the recurrence of cancer) or treat (e.g., manage orameliorate a cancer or one or more symptoms thereof) cancer. Any agentor therapy (e.g., chemotherapies, radiation therapies, targetedtherapies, such as imatinib, sorafenib and vemurafenib, hormonaltherapies, and/or biological therapies or immunotherapies) which isknown to be useful, or which has been used or is currently being usedfor the prevention, treatment, management or amelioration of cancer orone or more symptoms thereof can be used in combination with a compoundor composition of the invention described herein. Anti-cancer agents,but not limited to: 5-fluoruracil; acivicin; aldesleukin; altretamine;aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase;azacitidine; azetepa; azotomycin; batimastat; bicalutamide; bleomycinsulfate; brequinar sodium; bropirimine; busulfan; carboplatin;carmustine; carubicin hydrochloride; carzelesin; cedefingol;chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate;cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunorubicinhydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguaninemesylate; diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride;droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin;edatrexate; eflornithine hydrochloride; enloplatin; enpromate;epipropidine; epirubicin hydrochloride; erbulozole; esorubicinhydrochloride; estramustine; estramustine phosphate sodium; etanidazole;etoposide; etoposide phosphate; fazarabine; fenretinide; floxuridine;fludarabine phosphate; fluorouracil; flurocitabine; fosquidone;fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea;idarubicin hydrochloride; ifosfamide; ilmofosine; interleukin II(including recombinant interleukin II, or rIL2), interferon alpha-2a;interferon alpha-2b; interferon alpha-m; interferon alpha-n3; interferonbeta-I a; interferon gamma-I b; iproplatin; irinotecan hydrochloride;lanreotide acetate; letrozole; leuprolide acetate; liarozolehydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride;masoprocol; mechlorethamine hydrochloride; megestrol acetate;melengestrol acetate; melphalan; menogaril; mercaptopurine;methotrexate; methotrexate sodium; metoprine; meturedepa; mitomycin;mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid;nocodazole; ormaplatin; paclitaxel; pegaspargase; porfromycin;prednimustine; procarbazine hydrochloride; puromycin; rogletimide;safingol hydrochloride; semustine; simtrazene; sparfosate sodium;sparsomycin; spiromustine; spiroplatin; streptonigrin; streptozocin;sulofenur; talisomycin; tegafur; teloxantrone hydrochloride; temoporfin;teniposide; teroxirone; testolactone; thiamiprine; thioguanine;thiotepa; tiazofurin; tirapazamine; topotecan; trimetrexate;trimetrexate glucuronate; triptorelin; uracil mustard; uredepa;vapreotide; verteporfn; vinblastine sulfate; vincristine sulfate;vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate;vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate;vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicinhydrochloride. Targeted therapies include, but are not limited to,tyrosine kinase inhibitors (e.g., imatinib, sorafenib, and vemurafenib).The invention also encompasses administration of an anti-VISTA compoundof the invention in combination with radiation therapy comprising theuse of x-rays, gamma rays and other sources of radiation to destroy thecancer cells. Cancer treatments are known in the art and have beendescribed in such literature as the Physician's Desk Reference (57thed., 2003).

The anti-VISTA antibodies described herein are also useful, for example,in the treatment of chronic infectious diseases, such as HIV, HBV, HCV,and HSV, among others.

Various properties and sequence information for select anti-VISTAantibodies of the invention are provided in Tables 1A, 1B and 2 herein.

TABLE 1A CDR Sequences of Select Fully Human or Humanized anti-human VISTA antibodies Light- Heavy- chain VH Heavy-chain chainHeavy-chain Light-chain cdr2 Light-chain mAb ID family cdr1 (Imgt)cdr2 (Imgt) cdr3 (Imgt) cdr1 (Imgt) (Imgt) cdr3 (Imgt) VSTB50 B GYTFTNYGINPYTGEP AREGYGNYIFPY ESVDTYANSL RAS QQTNEDPRT (SEQ ID NO: 1) (SEQ ID(SEQ ID NO: 3) (SEQ ID NO: 4) (SEQ ID (SEQ ID NO: 6) NO: 2) NO: 5)VSTB53 GYTFTHYT IIPSSGYS ARGAYDDYYDYYAMD QTIVHSNGNTY KVS FQASHVPWT(SEQ ID NO: 7) (SEQ ID Y (SEQ ID NO: 10) (SEQ ID (SEQ ID NO: 12) NO: 8)(SEQ ID NO: 9) NO: 11) VSTB60 B GYTFTNYG INTYTGES ARDYYGIYVSAYESVDNYANSF RAS QQSHEDPYT (SEQ ID (SEQ ID (SEQ ID NO: 15) (SEQ ID NO: 16)(SEQ ID (SEQ ID NO: 13) NO: 14) NO: 17) NO: 18) VSTB95 GFTFRNYG IISGGSYTARIYDHDGDYYAMDY QSIVHSNGNTY KVS FQGSHVPWT (SEQ ID (SEQ ID(SEQ ID NO: 21) (SEQ ID NO: 22) (SEQ ID (SEQ ID NO: 19) NO: 20) NO: 23)NO: 24) VSTB112 D GGTFSSYA IIPIFGTA ARSSYGWSYEFDY QSIDTR SAS OQSAYNPIT(SEQ ID (SEQ ID (SEQ ID NO: 27) (SEQ ID NO: 28) (SEQ ID (SEQ ID NO: 25)NO: 26) NO: 29) NO: 30) VSTB116 D GGTFSSYA IIPIFGTA(SEQ ARSSYGWSYEFDYQSINTN AAS QQARDTPIT (SEQ ID ID (SEQ ID NO: 33) (SEQ ID NO: 34) (SEQ ID(SEQ ID NO: 36) NO: 31) NO: 32) NO: 35)

TABLE 1B Heavy and Light Chain Sequences of Select Fully Human orHumanized anti-human VISTA antibodies Protein ID Heavy-chain AA CDSLight-chain AA CDS VSTB50QVQLVQSGSELKKPGASVKVSCKASGYTFTNYGLNWVRQAPGQGLEWDIVMTQTPLSLSVTPGQPASISCRASESVDTMGWINPYTGEPTYADDFKGRFVFSLDTSVSTAYLQICSLKAEDTAVYYCAYANSLMHWYLQKPGQPPQLLIYRASNLESREGYGNYIFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLGVPDRFSGSGSGTDFTLKISRVEAEDVGVYVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTYCQQTNEDPRTFGQGTKLEIKRTVAAPSVFQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPIFPPSDEQLKSGTASVVCLLNNFYPREAKVQKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEWKVDNALQSGNSQESVTEQDSKDSTYSLSQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRSTLTLSKADYEKHKVYACEVTHQGLSSPVTKEPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTSFNRGEC (SEQ ID NO: 48)PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK (SEQ ID NO: 47)VSTB53 QVQLVQSGAEVKKPGASVKVSCKASGYTFTHYTIHWVRQAPGQGLEWDIVMTQSPLSLPVTAGEPASISCRSSQTIVHMGYIIPSSGYSEYNQKFKDRVTMTRDTSTSIVYMELSSLRSEDTAVYYCASNGNTYLEWYLQKPGQSPQLLIYKVSNRFSRGAYDDYYDYYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAGVPDRFSGSGSGTDFTLKISRVEAEDVGVYALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSLSSVVTVPSYCFQASHVPWTFGQGTKLEIKRTVAAPSVFSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVIFPPSDEQLKSGTASVVCLLNNFYPREAKVQFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTWKVDNALQSGNSQESVTEQDSKDSTYSLSKPREEQYNSTYRVVSVONLHQDWLNGKEYKCKVSNKALPAPIEKTISKASTLTLSKADYEKHKVYACEVTHQGLSSPVTKKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPESFNRGEC (SEQ ID NO: 50) NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 49) VSTB60QVQLVQSGSELKKPGASVKVSCKASGYTFTNYGMTWVRQAPGQGLEWDIVMTQTPLSLSVTPGQPASISCRASESVDMGWINTYTGESTYADDFKGRFVFSLDTSVSTAYLQICSLKAEDTAVYYCANYANSFMHWYLQKPGQSPQLLIYRASNLERDYYGIYVSAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLSGVPDRFSGSGSGTDFTLKISRVEAEDVGVVKDYFPEPVTVSWNSGALTSGVFITFPAVLQSSGLYSLSSVVTVPSSSLGTYYCQQSHEDPYTFGQGTKLEIKRTVAAPSVQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPFIFPPSDEQLKSGTASVVCLLNNFYPREAKVKPKDILMISRTPEVTCVVVOVSHEDPEVKFNWYVDGVEVHNAKTKPREEQWKVDNALQSGNSQESVTEQDSKDSTYSLQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRSSTLTLSKADYEKHKVYACEVTHQGLSSPVTEPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTKSFNRGEC (SEQ ID NO: 52)PPVLDSOGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSISL SPGK (SEQ ID NO: 51)VSTB95 EVQLVESGGGLVQPGGSLRLSCAASGFTFRNYGMSWVRQAPGKGLEWDIVMTQSPLSLPVTPGEPASISCRSSQSIVHVASIISGGSYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSIYDHDGDYYAMDYWGQGTIVIVSSASTKGPSVFPLAPSSKSTSGGTAALGVPDRFSGSGSGTDFTLKISRVEAEDVGVYGCLVKDYFPEPVIVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLYCFQGSHVPWTFGQGTKLEIKRTVAAPSVFGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFIFPPSDEQLKSGTASVVCILNNFYPREAKVQPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPWKVDNALQSGNSQESVTEQDSKDSTYSLSREEQYNSTYRVVSVLTVLHQDWINGKEYKCKVSNKALPAPIEKTISKAKGSTLTLSKADYEKHKVYACEVTHQGLSSPVTKQPREPQVYTIPPSRDELTKNQVSLICLVKGFYPSDIAVEWESNGQPENNSFNRGEC (SEQ ID NO: 54) YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 53) VSTB112QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWDIQMTQSPSSLSASVGDRVTITCRASQSIDTMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARRLNWYQQKPGKAPKLLIYSASSLQSGVPSRSSYGWSYEFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLFSGSGSGTDFTLTISSLQPEDFATYYCQQSAVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTYNPITEGQGTKVEIKRTVAAPSVFIFPPSDEQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPQLKSGTASVVCLLNNFYPREAKVQWKVDNKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEALQSGNSQESVTEQDSKDSTYSLSSTLTLSKQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRADYEKHKVYACEVTHQGLSSPVTKSFNRGEEPQVYTLPPSRDELTKNQVSLICLVKGFYPSDIAVEWESNGQPENNYKTT C (SEQ ID NO: 56)PPVLDSDGSFELYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK (SEQ ID NO: 55)VSTB116 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWDIQMTQSPSSLSASVGDRVTITCRASQSINTMGGIIPIEGTANYAQKFQGRVTITADESTSTAYMELSSIRSEDTAVYYCARNLNWYQQKPGKAPKLLIYAASSLQSGVPSRSSYGWSYEFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLFSGSGSGTDFTLTISSLOPEDFATYYCQQARVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTDTPITFGQGTKVEIKRTVAAPSVFIFPPSDEQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPQLKSGTASVVCLLNNFYPREAKVQWKVDNKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEALQSGNSQESVTEQDSKDSTYSLSSILTLSKQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRADYEKHKVYACEVTHQGLSSPVTKSFNRGEEPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT C (SEQ ID NO: 58)PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK (SEQ ID NO: 57)VSTB140* QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWDIQMTQSPSSLSASVGDRVTITCRASQSIDTMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARRLNWYQQKPGKAPKWYSASSLQSGVPSRSSYGWSYEFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLFSGSGSGTDETLTISSLOPEDFATYYCQQSAVKDYFPEPVTVSWNSGALTSGVHTFPAVLCISSGLYSLSSVVIVPSSNFGTYNPITFGQGTKVEIKRTVAAPSVFIFPPSDEQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPAAASSVELFPPKPKDQLKSGTASVVCLLNNFYPREAKVQWKVDNTLMISRTPEVTCVVVDVSAEDPEVQFNWYVDGVEVHNAKTKPREEQFNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKSTFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQADYEKHKVYACEVTHQGLSSPVTKSFNRGEVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP C (SEQ ID NO: 56)MLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK (SEQ ID NO: 59)VSTB149*^(Δ) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWDIQMTQSPSSLSASVGDRVTITCRASQSIDTMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARRLNWYQQKPGKAPKLLIYSASSLQSGVPSRSSYGWSYEFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLFSGSGSGTOFTLTISSLOPEDFATYYCQQSAVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTYNPITFGQGTKVEIKRTVAAPSVFIFPPSDEQTYICNVNHKPSNTINDKKVEPKSCDKTHTCPPCPAPPVAGPDVFLFPPQLKSGTASVVCLLNNFYPREAKVQWKVDNKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEALQSGNSQESVTEQDSKDSTYSLSSTLTLSKQYNSTYRVV5VLTVLHQDWLNGKEYKCKVSNAALPAPIAKTISKAKGQPADYEKHKVYACEVTHQGLSSPVTKSFNRGEREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK C (SEQ ID NO: 56)TIPPVLDSDGSFFLYSKLIVOKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK (SEQ ID NO: 60)VSTB174* QVQLVQSGAEVKKPGSSVKVSCKASGGTESSYAISWVRQAPGQGLEWDIQMTQSPSSLSASVGDRVTITCRASQSIDT MGGIIP1FGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARRLNWYQQKPGKAPKLLIYSASSLQSGVPSRSSYGWSYEFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLFSGSGSGTDFTLTISSLQPEDFATYYCQQSAVKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSLSSVVTVPSSSLGTYNPITEGQGTKVEIKRTVAAPSVFIFPPSDEQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLEPPQLKSGTASVVCLLNNFYPREAKVQWKVDNKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEALQSGNSQESVTEQDSKDSTYSLSSTLTLSKQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRADYEKHKVYACEVTHQGLSSPVTKSENRGEEPQVYTLPPSREEMTKNQVSLICLVKGFYPSDIAVEWESNGQPENNYKT C (SEQ ID NO: 56)TPPVLDSDGSFELYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLS LSPGK (SEQ ID NO: 61)*Constant region sequences in VSTB140, VSTB149 and VSTB174 areunderlined. ^(Δ)Amino acid residues conferring protease resistance inthe heavy chain of VSTB149 are indicated in bold.

TABLE 2 Dissociation constant (K_(D)) for select anti-VISTA antibodiesSample KD (M) ka1 (1/Ms) kd1 (1/s) S1 1.71E−10 1.69E+06 2.89E−041.09E−10 1.11E+06 1.21E−04 S40 5.07E−10 1.46E+05 7.40E−05 6.96E−101.39E+05 9.69E−05 S41 6.32E−10 4.82E+05 3.05E−04 3.10E−10 7.08E+052.19E−04 S42 1.04E−10 1.05E+06 1.09E−04 2.65E−10 5.13E+05 1.36E−04 S432.64E−11 1.25E+06 3.30E−05 5.28E−11 1.18E+06 6.22E−05 S44 2.53E−111.23E+06 3.12E−05 6.40E−11 9.93E+05 6.36E−05 S45 2.35E−11 1.58E+063.72E−05 2.58E−11 1.46E+06 3.77E−05 S46 1.06E−10 1.56E+06 1.66E−042.96E−10 1.50E+06 4.44E−04 S47 3.56E−10 5.14E+05 1.83E−04 2.52E−105.69E+05 1.43E−04 S33 8.30E−10 1.23E+06 1.02E−03 1.22E−09 8.96E+051.10E−03 S34 1.08E−09 5.95E+05 6.43E−04 2.80E−09 5.20E+05 1.46E−03 S358.06E−11 2.08E+06 1.68E−04 1.35E−10 1.78E+06 2.41E−04 S36 6.29E−111.77E+06 1.12E−04 2.90E−11 1.58E+06 4.58E−05 S37 2.23E−09 5.10E+051.14E−03 4.43E−09 3.94E+05 1.75E−03 S38 2.26E−09 5.18E+05 1.17E−032.03E−09 5.37E+05 1.09E−03 S39 5.62E−10 3.97E+05 2.23E−04 3.47E−104.15E+05 1.44E−04 S25 1.31E−09 6.21E+05 8.12E−04 1.10E−09 5.65E+056.24E−04 S26 No Binding 3.53E−09 2.38E+05 8.41E−04 S27 1.13E−09 8.86E+059.97E−04 1.61E−09 7.12E+05 1.15E−03 S48 3.12E−10 1.24E+06 3.87E−041.21E−09 8.78E+05 1.06E−03 S28 2.03E−09 1.08E+06 2.19E−03 2.03E−099.30E+05 1.88E−03 S29 3.78E−11 1.42E+06 5.38E−05 8.90E−11 9.06E+058.06E−05 S30 No Binding No Binding S31 Weak Binding Weak Binding S32Weak Binding Weak Binding S15 9.34E−11 6.46E+05 6.04E−05 5.13E−103.50E+05 1.80E−04 S16 1.26E−10 5.54E+05 6.99E−05 1.92E−10 4.43E+058.53E−05 S17 7.68E−10 9.88E+05 7.59E−04 4.10E−10 7.09E+05 2.91E−04 S182.28E−09 4.90E+05 1.12E−03 1.05E−09 3.13E+05 3.29E−04 S19 1.54E−091.02E+06 1.58E−03 2.86E−10 7.03E+05 2.01E−04 S20 1.48E−09 6.67E+059.85E−04 4.57E−10 6.36E+05 2.91E−04 S21 3.18E−09 3.16E+05 1.00E−031.34E−09 2.70E+05 3.60E−04 S22 2.98E−09 1.09E+06 3.25E−03 1.27E−091.25E+06 1.59E−03 S6 6.36E−10 5.28E+05 3.36E−04 3.02E−10 5.98E+051.80E−04 S7 6.75E−10 1.31E+06 8.87E−04 3.27E−10 1.15E+06 3.75E−04 S81.15E−10 1.89E+06 2.18E−04 5.97E−11 1.25E+06 7.48E−05 S9 1.67E−101.87E+06 3.11E−04 9.31E−11 1.27E+06 1.18E−04 S10 8.90E−11 1.55E+061.38E−04 4.30E−11 1.22E+06 5.27E−05 S12 4.94E−10 1.57E+06 7.76E−042.39E−10 1.19E+06 2.86E−04 S13 1.02E−10 1.42E+06 1.44E−04 6.46E−119.55E+05 6.17E−05 S14 2.02E−10 1.26E+06 2.55E−04 7.55E−11 1.12E+068.43E−05 S1 2.06E−10 1.60E+06 3.29E−04 8.35E−11 1.21E+06 1.01E−04 S21.56E−10 9.74E+05 1.52E−04 8.66E−11 7.25E+05 6.28E−05 S3 4.33E−119.07E+05 3.93E−05 4.89E−11 7.41E+05 3.63E−05 S4 1.52E−10 8.98E+051.36E−04 7.54E−11 6.93E+05 5.23E−05 S49 1.45E−10 1.01E+06 1.46E−041.04E−10 7.28E+05 7.60E−05 S5 2.13E−10 1.25E+06 2.67E−04 1.37E−108.51E+05 1.17E−04

EXAMPLES Example 1: Analysis of Vista Expression on Human HematopoieticCells

Methods:

Preparation and Staining of Fresh Human PBMCs for VISTA Expression

Expression of VISTA was tested on freshly isolated human PBMCs(peripheral blood mononuclear cells) from several donors. Anti-HumanVISTA-biotin (GA-1) was used for staining (5 μg/ml). Mouse IgG1,K-biotin (Clone MOPC-21 at 5 μg/ml) was used as an isotype control.

Donor Material

Blood samples were obtained from Biological Specialty Corp. (Colmar,Pa.) and were collected and analyzed the same day. 10 ml of whole bloodcontaining heparin sulfate were couriered for analysis.

Sample Preparation

Blood was diluted 1:1 in sterile PBS. 22 ml diluted cord blood waslayered onto 20 ml sterile Ficoll-Hypaque (GE Healthcare Cat #17-144003)in 50 ml conical tubes. Tubes were centrifuged at 1800 rpm for 20minutes at room temperature. Mononuclear cells at the interfacefollowing centrifugation were harvested using a 1 ml pipettor andcombined into two 50 ml conical tubes. Sterile PBS was added to eachtube to make the volume up to 50 ml and the cells were centrifuged at300 g for 10 minutes at 4° C. Supernatant was discarded. Cells wereresuspended in 50 ml of sterile PBS and tubes were spun at 300 g for 10minutes at 4° C. Supernatant was discarded. Cells were combined andresuspended in 50 ml sterile PBS prior to counting.

Staining Protocol: A frozen vial containing 5×10⁷ PBMCs was used forcompensation controls and as a control for staining.

The following reagents and/or consumables were used:

FACS Stain Buffer (BSA) from BD Biosciences (Cat #554657) supplementedwith 0.2% EDTA; Phosphate-Buffered saline (PBS) (Gibco cat #14190);96-well polypropylene round-bottomed plate (BD #3077); 1.2 mlpolypropylene cluster tubes (Corning #4451); biotinylated Anti-VISTAclone GA-1 from ImmunoNext Lot #080612B (used at 5 μg/ml); biotinylatedmIgG1, K isotype control (Clone MOPC-21); Biolegend cat #400104, Lot#B116649 (used at 5 μg/ml); anti-human antibodies (See staining tablebelow); near-Infrared live/dead dye (Invitrogen, cat #L10119); andstreptavidin reagents including STP-APC (BD Biosciences cat #554067, Lot#04251) (used at 1:200 dilution in FACS buffer), STP-PE (Biolegend cat#405203, Lot #B139688) (used at 1:200 dilution in FACS buffer), STP-PECy7 (showed non-specific binding in isotype control samples), STP-Q605(Invitrogen cat #Q10101MP, Lot #53449A) (used at 1:200 dilution in FACSbuffer).

Cell Surface Staining Protocol

Prior to staining, 1×10⁶ cells were transferred into 96-wellround-bottomed plates and were washed with 150 μl PBS. Plates were thencentrifuged at 1300 rpm at 4° C. for 3 minutes.

Subsequently, cells were washed again in PBS and centrifuged asdescribed above.

Live/dead staining was then performed in 50 μl PBS containing 0.25 μl ofnear-infrared live/dead dye. After 10 minutes at room temperature thewells were washed with 150 μl FACs staining buffer and centrifuged at1300 rpm at 4° C. for 3 minutes. Supernatant was discarded.

Cells were blocked with human serum at 1:100 in 50 μl FACS stainingbuffer. Plates were incubated at 4° C. for 15 minutes. Wells were thenwashed with 150 μl FACs staining buffer and centrifuged at 1300 rpm at4° C. for 3 minutes. Supernatant was discarded.

A cocktail containing the following antibodies was then added to eachwell for surface staining: The cocktails are described in Tables 3-6below. Each cocktail would be utilized separately from the othersdepending on the populations of interest.

TABLE 3 Lineage Stain Titer Target (μl/10⁶ Fluoro Antigen Mouse RatHuman Isotype Clone Supplier Cat No. Lot No. Cells) FITC/AF488 CD19 XmIgG1 HIB19 Biolegend 302206 B123019 2 PE CD11b X mIgG1, K ICRF44 BDBio. 555388 45134 2 PerCP-Cy5.5 HLA-DR X mIgG2a, K G46-6 BD Bio. 56065225161 0.5 PE Cy7 CD16 X mIgG1, K 3G8 BD Bio. 557744 87825 0.2 APC Cy7NIR X Live/Dead AF700 CD56 X mIgG1, K B159 BD Bio. 557919 19470 1APC/AF647 VISTA-Bio X PB/V450 CD3 X mIgG1, K UCHT1 BD Bio. 558117 909260.5 Q605 CD14 X mIgG2a, K TuK4 Invitrogen Q10013 1049158 0.2

TABLE 4 T Cell Stain Titer Target (μl/10⁶ Fluoro Antigen Mouse Rat HumanIsotype Clone Supplier Cat No. Lot No. Cells) FITC/AF488 CD4 X mIgG1, KRPA-T4 BD Bio. 555346 38460 2 PE VISTA-Bio X PerCP-Cy5.5 CD8 X mIgG1, KRPA-T8 BD Bio. 560662 1037 0.5 PE Cy7 CD56 X mIgG1, K B159 BD Bio.557747 47968 0.5 APC Cy7 NIR X AF700 CD45RO X mIgG2a, K UCHL1 Biolegend304218 B143062 1 APC/AF647 TCRgd X mIgG, K B1 Biolegend 331212 B126473 2PB/V450 CD45RA X mIgG2b, K HI100 BD Bio. 560363 90928 0.5 Q655 CD3 XmIgG2a S4.1 Invitrogen Q10012 982352 0.5

TABLE 5 DC Stain Titer Target (μl/10⁶ Fluoro Antigen Mouse Rat HumanIsotype Clone Supplier Cat No. Lot No. Cells) FITC/AF488 Lin1 X Mix MixBD Bio. 340546 2152758 5 PE CD11c X mIgG1, K BD Bio. 555392 45123 2PerCP-Cy5.5 HLA-DR X mIgG2a, K G46-6 BD Bio. 560652 25161 0.5 APC Cy7NIR X APC/AF647 CD83 X mIgG1, K HB15e BD Bio. 551073 57688 2 BV421 CD123X mIgG1, K 6H6 Biolegend 306017 B148193 0.5 Q605 VISTA-Bio X

TABLE 6 Myeloid Stain Titer Target (μl/10⁶ Fluoro Antigen Mouse RatHuman Isotype Clone Supplier Cat No. Lot No. Cells) FITC/AF488 CD33 XmIgG1 HM3-4 Biolegend 303304 B100963 3 PE CD11b X mIgG1, K ICRF44 BDBio. 555388 45134 2 APC Cy7 NIR X APC/AF647 VISTA-Bio X Q605 CD45 XmIgG1, K HI30 Invitrogen Q10051 880470 1

Following the surface staining, cells were washed twice as previouslydescribed with FACS staining buffer and centrifuged at 1300 rpm at 4° C.for 5 minutes. Samples were resuspended in 50 μl of FACS staining buffercontaining the appropriate fluorescently-labeled streptavidin. Sampleswere incubated at 4° C. for 30 minutes. Cells were washed with 150 μlFACS staining buffer and centrifuged at 1300 rpm at 4° C. for 5 minutes.This wash step was repeated before samples were resuspended in 250 μl ofFACS staining buffer. Samples were analyzed on a BD LSRFortessa™ cellanalyzer (BD Biosciences) the same day.

Data Analysis

Flow cytometry data was reanalyzed using FlowJo Version 9 software togate specific phenotypic populations. Enumeration of geometric mean wasused to compare VISTA expression in different cell subsets. Eachpopulation was normalized for background by subtracting isotype controlvalues from the mean values of the anti-VISTA treated samples. Graphswere prepared in Prism and statistics were performed using eitherstudent's T-test if only two samples were compared, or one-way ANOVAwith Bonferroni post-tests.

Results:

Expression of VISTA on Human Myeloid and Lymphoid Subsets:

As shown in FIGS. 2A-2E, 3A-3G, 4, 5A-5B and 6A-6C, VISTA expression onCD14⁺ monocytes was significantly different from all other populations(p<0.001). No significant differences between other populations wereseen. Monocytes expressed the highest levels of VISTA in peripheralblood, with the CD14⁺CD16⁻ subset having significantly higher expressionthan CD14^(lo) CD16⁺ cells. While APCs showed moderate expression ofVISTA, lymphoid subsets showed low expression levels.

Expression of VISTA on Human T and NK Subsets:

As shown in FIGS. 7A-7E, 8A-8G and 9, with NK subsets, CD56^(lo) cellsexhibited significantly higher expression levels of VISTA than CD56^(Hi)NK cells. Of T cell subsets, CD8⁺ memory cells expressed the highestexpression levels, although they are not significantly higher than CD8⁺naive or CD4⁺ T cells.

Expression of VISTA on Human Dendritic Cell Subsets:

As shown in FIGS. 10A-10D, 11A-11C and 12, no significant differences inVISTA expression seen; DCs and basophils exhibited low expression ofVISTA, with plasmacytoid dendritic cells (pDCs) generally being higherbut not to a significant extent.

Conclusion: These results show expression of VISTA on various immunecell subsets, and that VISTA is expressed on monocytes most highly, withsome expression on different T cell subsets and NK cells, and little tono expression on B cells.

Example 2: Vista Expression on Peripheral Blood Cells

Methods:

Staining of whole blood: Freshly isolated whole blood (100 μl) wasstained with antibody cocktails as indicated below by incubation for 30minutes at 4° C. Red blood cells (RBCs) were lysed with RBC lysis bufferand the remaining cells were washed 1× with staining buffer. Cells werere-suspended in 200 μl of staining buffer. The data were collected usinga MACSQuant flow cytometer and analyzed using FlowJo analysis software.

Staining of peripheral blood mononuclear cells (PBMCs): Peripheral bloodmononuclear cells were isolated from whole blood using Ficoll gradient.Freshly isolated 1×10⁶ PBMCs were stained with antibody cocktails in 100μl of staining buffer. Samples were incubated for 30 minutes at 4° C.then washed once with staining buffer. Cells were re-suspended in 100 μlof staining buffer. The data were collected using MACSQuant® flowcytometer (Miltenyi Biotec) and analyzed using FlowJo analysis software.

The antibodies used were CD11b, CD33, CD177, CD16, CD15, CD14, CD20,HLADR, CD3, CD4, CD8, CD127, CD69, and FOXP3 antibodies (Biolegend, SanDiego, Calif.). The APC-conjugated mouse anti-human VISTA (clone GG8)was made by ImmuNext (Lebanon, N.H.).

Conclusions:

Expression of VISTA on healthy human peripheral blood cells

Whole blood and peripheral blood mononuclear cells were analyzed forVISTA expression using multicolor flow cytometry. As shown in FIGS. 15Aand 15B, the highest level of VISTA expression was detected on monocytesfollowed by neutrophils. Both the CD4+ and CD8+ T cells expressed lowlevel of VISTA as shown in FIGS. 13C and 13D.

Expression of VISTA on Cancer Patient Peripheral Blood Cells

As shown in FIGS. 14A-C, peripheral blood mononuclear cells (PBMCs) fromlung cancer patients were analyzed. FIG. 14A is a representative flowplot showing analysis of CD14⁺ monocytes and CD15⁺ myeloid derivedsuppressive cells (MDSCs). The results suggest that phenotypically CD15⁺cells are neutrophil derived MDSCs. Additionally, these cells are absentin healthy blood samples. FIG. 14B is a representative histogram ofVISTA expression on healthy and cancer patient derived monocytes,suggesting a higher level of VISTA expression on cancer patient cellscompared to healthy controls. Similarly higher level of VISTA was foundon MDSCs in cancer patients, as shown in FIG. 14C.

FIG. 15A is a representative FACS plot showing the presence ofneutrophil derived MDSCs in the blood of colon cancer patients. FIGS.15B and 15C are representative histograms showing higher level of VISTAexpression on cancer patients' monocytes compare to healthy donor bloodsamples.

Expression of VISTA on Cynomolgus Monkey Peripheral Blood Cells

As shown in FIGS. 16A and 16B flow cytometry analysis of monkey wholeblood revealed the VISTA expression pattern similar to human cells. Bothmonocytes and neutrophils expressed the highest level of VISTA comparedto CD4⁺ (FIG. 16C) and CD8⁺ (FIG. 16D) T cells.

Example 3: Vista Expression in Heme Malignancy Cell Lines at the RNALevel and Protein Level

Because VISTA is expressed in heme malignancies, an anti-VISTA antibodycould potentially target the malignant cells for destruction, as well asblock VISTA and promote anti-tumor immune responses.

The data includes RNAseq analysis of ˜140 heme malignancy cell lines(some cell lines are repeated in the analysis). The data is shown inFIG. 17.

The RNAseq values are listed as FPKM (Fragments Per Kilobase of exon perMillion fragments mapped) values.

In essence, this means that all reads falling in the exonic regions of agene were counted and normalized by both the length of the gene and thetotal number of reads per sample (to account for inter-sampledifferences). The cutoff value is 1; above 1 is positive for VISTAexpression (at the RNA level), below 1 is negative for VISTA expression.

The results indicated that many cell lines are positive at the RNAlevel, primarily acute myeloid leukemias and chronic myelogenousleukemias. This may be expected since VISTA is highly expressed innormal myeloid cells, and because its function is believed to dampenimmune responses, including anti-tumor immune responses.

Example 4: Generation of Monoclonal Antibodies Against Vista

Phage Panning

Twenty four phage panning experiments were carried out to enrich forphage reactive to Cyno VISTA-His. The cynomolgus VISTA protein was usedfor these experiments as it showed better biotin conjugation than thehuman VISTA protein. To determine the success of the phage experiments,phage pools from the individual panning rounds were added to neutravidinplates coated with biotinylated cyno VISTA-His and detected with aHRP-conjugated anti-M13 antibody. Individual colonies were picked fromthe phage selection rounds and Fabs proteins were produced in 96 wellplates. The expressed Fab supernatants were assayed for binding tobiotinylated cyno VISTA-His. This resulted in more than 200 hits.

The VH and VL regions from the Fab plates were amplified, submitted forDNA sequencing and were exported as FASTA files. When picking the clonesthat should be converted and tested as MABs, the clones were chosenbased on sequence diversity as well as having limited post-translationalmodification risks and as few hydrophobic residues as possible.

The VH and VL from the phage clones were sub-cloned into mammalianIgG1/kappa expression vectors and transfected into HEK293 cells. Theantibodies were purified on Protein A Sepharose Fast Flow affinityresin. The concentration of the phage MABs was determined byquantitative ELISA using Nanodrop measurements, The antibody panel wasexpressed at high levels. SDS-PAGE analysis demonstrated the integrityof each expressed antibody variant.

In-line maturation of the phage antibodies was done by amplifying the VHdomains from the polyclonal antibody mixes from the last round ofpanning for cloning into phage vectors that have diversity in the VL.This resulted in an enriched VH pool which was sampled with additionaldiversity in the VL. The phage were taken through 1-2 rounds ofstringent panning with the expectation to identify very high affinitybinders to VISTA ECD His protein. A monoclonal Fab ELISA was run todetermine the success of the maturation. ELISA and expression data wasnormalized to a reference clone set to 100% from the original de novopalming experiment and affinity matured clones with higher bindingsignal to cyno VISTA antigen than the reference clone were identified.This process generated several clones that demonstrated up to 200%binding when screened at low antigen concentration (1 nM), the cloneswith highest affinity were sequenced and produced as MABs.

Hybridoma Generation

One group of BALB/cAnNCrl mice received one intraperitoneal (IP)injection of 50 μg Hu VISTA-Ig recombinant protein (Sino) emulsified inComplete Freund's Adjuvant followed two weeks later by one IP injectionof 50 μg Hu VISTA-Ig recombinant protein emulsified in IncompleteFreund's Adjuvant. Two weeks later the mice received one IP injection of50 μg cyno VISTA-Fc recombinant protein emulsified in IncompleteFreund's Adjuvant. All mice received a final injection of 25 μg humanand 25 μg cyno VISTA at the base of tail in PBS, five days prior tosplenic harvest for fusion.

Another group of BALB/cAnNCrl mice received one IP injection of 50 μg HuVISTA-His recombinant protein emulsified in Complete Freund's Adjuvant.Two weeks later the mice received one IP injection of 50 μg Hu VISTA-Hisrecombinant protein emulsified in Incomplete Freund's Adjuvant. Twoweeks later the mice received one IP injection of 50 μg Cyno VISTA-Hisrecombinant protein emulsified in Incomplete Freund's Adjuvant. Twoweeks later all mice received a final injection of 25 μg Hu VISTA-Hisand 25 μg Cyno VISTA-His in PBS, three days prior to splenic harvest forfusion.

On the day of fusion, mice were euthanized by CO2 asphyxiation, thespleens were removed and placed into 10 mL of cold phosphate-bufferedsaline. A single cell suspension of splenocytes was prepared by grindingspleens through a fine mesh screen with a small pestle and rinsing withPBS at room temperature. Cells were washed once in PBS and subjected toRBC lysis. Briefly, cells were resuspended in 3 mL of RBC lysis buffer(Sigma #R7757) per every spleen and placed on ice for 5 minutes. Cellswere again washed once in PBS at room temperature and labeled formagnetic sorting. As per manufacturer's instructions, cells were labeledwith anti-murine Thy1.2, anti-murine CD11b and anti-murine IgM magneticbeads (Miltenyi Biotec #130-049-101, 130-049-601 and 130-047-301respectively) then sorted using a MS column with a Midi MACS. Thenegative cell fractions (positive cell fractions were discarded) werefused to FO cells. Fusion was carried out at a 1:1 ratio of murinemyeloma cells to viable spleen cells. Briefly, spleen and myeloma cellswere mixed together, pelleted and washed once in 50 mL of PBS. Thepellet was resuspended with 1 mL of polyethylene glycol (PEG) solution(2 g PEG molecular weight 4000, 2 mL DMEM, 0.4 mL DMSO) per 10e8splenocytes at 37° C. for 30 seconds. The cell/fusion mixture was thenimmersed in a 37° C. water bath for approximately 60 seconds with gentleagitation. The fusion reaction was stopped by slowly adding 37° C. DMEMover 1 minute. The fused cells were allowed to rest for 5 minutes atroom temperature and then centrifuged at 150×g for 5 minutes. Cells werethen resuspended in Medium E-HAT (MediumE (StemCell Technologies cat#03805) containing HAT (Sigma cat #H0262) and seeded in 96-well flatbottom polystyrene tissue culture plates (Corning #3997).

A capture EIA was used to screen hybridoma supernatants for antibodiesspecific for cyno VISTA. Briefly, plates (Nunc-Maxisorp #446612) werecoated at 4 μg/ml for at least 60 minutes with goat anti-mouse IgG (Fc)antibody (Jackson #115-006-071) in coating buffer (Thermo 28382). Plateswere blocked with 200 μl/well of 0.4% (w/v) bovine serum albumin (BSA)in PBS at for 30 minutes at RT. Plates were washed once and 50 μl/wellof hybridoma supernatant was added and incubated at room temperature forat least 30 minutes. Plates were washed once and 50 μl/well of 0.1 μg/mLof cyno VISTA-hulg was added and incubated at RT for 30 minutes. Plateswere washed once and 1:40,000 Streptavidin HRP (Jackson 016-030-084) in0.4% BSA/PBS was added to plates and incubated for 30 minutes at RT.Plates were washed 3× and subsequently developed using 100 μl/well TMBTurbo substrate (Thermo Scientific 34022) incubating approximately 10minutes at RT. The reaction was stopped using 25 μl/well 4N SulfuricAcid and absorbance was measured at 450 nm using an automated platespectrophotometer. Fifteen of the primary hits were selected forsubcloning by limiting dilution and were screened in the same primaryscreen format.

All cyno VISTA reactive hybridoma cell lines were cross screened usinghuman VISTA-Ig to assess cross-reactivity. Briefly, plates(Nunc-Maxisorp #446612) were coated at 4 μg/mL with goat anti-ms Fc(Jackson #115-006-071) in 0.1M sodium carbonate-bicarbonate buffer, pH9.4 (Pierce 28382 BupH™) 0/N at 4° C. Without washing, the wells wereblocked with 200 μl of block (0.4% BSA (Sigma) (w/v) in PBS(Invitrogen)) overnight at 4° C. After removing block solution,undiluted hybridoma supernatants were incubated on coated plates for 30minutes at RT. Plates were washed once with PBST (0.02% Tween 20 (Sigma)(w/v) in PBS), and then incubated for 30 minutes with Hu VISTA-Igdiluted to 100 ng/ml in block. Plates were washed once with and probedwith Goat antihuman-Fc-HRP (Jackson #109-036-098) diluted 1:10,000 inblock for 30 minutes at RT. Plates were again washed and subsequentlydeveloped using 1000/well TMB Turbo substrate (Thermo Scientific 34022)incubating approximately 10 minutes at RT. The reaction was stoppedusing 25 μl/well 4N Sulfuric Acid and absorbance was measured at 450 nmusing an automated plate spectrophotometer.

Hybridomas that were shown to be reactive to both human and cynomolgusVISTA had their V region antibody sequences cloned. Hybridoma cells wereprepared prior to the reverse transcriptase (RT) reactions withInvitrogen's SuperScript III cells Direct cDNA System. Briefly, theculture medium was discarded and the plate placed on ice and resuspendedin 200 μl cold PBS. Forty microliters was transferred to a MicroAmp fast96 well Reaction PCR plate and the plate was placed on a cold metalplate base, sealed with plastic film and spun at 700 rpm for 3 minutes.The PBS was discarded and to each well, 10 μl Resuspension Buffer and 1μl Lysis Enhancer was added. The plate was sealed and incubated at 75°C. for 10 min and stored at −80° C.

For the RT reaction, each well contained 5 μl water, 1.6 μl 10× DNaseBuffer, 1.2 μl 50 mM EDTA, 2 μl Oligo(dT)20 (50 mM) and 1 μl 10 mM dNTPMix. The plate was incubated at 70° C. for 5 min, followed by incubationon ice for 2 min, then the following reagents were added for each well;6 μl 5×RT Buffer, 1 μl RNaseOUT™ (40 U/μl), 1 μl SuperScript™ III RT(200 U/μl) and 1 μl of 0.1M DTT. The plate was sealed and placed on athermal cycler preheated to 50° C. and incubated at 50° C. for 50minutes, followed by inactivation (5 min incubation at 85° C.). Thereaction was chilled on ice and the single-stranded cDNA was stored at−80° C. until further use.

For V region amplifications, 20 μl PCR reactions were set up. Each wellcontained 16.2 μl water, 2.0 μl 10×PCR Reaction buffer, 0.8 μl MgSO4 (50mM), 0.4 μl 10 mM dNTP, 0.15 μl 100 uM Forward primer mix 0.05 μl 100 uMReverse primer, 0.2 μl HiFi Tag enzyme. The cDNA, prepared as describedabove, was transferred (2 μl/well) to the PCR components mixture, theplate was sealed and an amplification reaction was run; for VH theprogram was (i) 94° C. for 1 min (ii) 94° C. for 15 sec (iii) 55° C. for30 sec (iv) 68° C. for 1 min. Steps (ii-iv) were repeated for a total of35 cycles followed by a final extension at 68° C. for 3 min. for VL theprogram was (i) 94° C. for 1 min (ii) 94° C. for 15 sec (iii) 55° C. for30 sec (iv) 65° C. for 30 sec, (v) 68° C. for 1 min. Steps (ii-v) wererepeated for a total of 35 cycles followed by a final extension at 68°C. for 3 min.

Forward primers were pre-mixed and such mixture was used in ration 3:1with the reverse primer. PCR products were verified on an agarose gel.The reactions were prepared for infusion cloning by the addition ofEnhancer (In-Fusion HC Cloning Kit, cat #639650, Clontech). Fivemicroliters of the PCR reaction was transferred to a PCR plate followedby the transfer of 2 μl of enhancer/well. The plate was sealed andincubated in a thermal cycler (15 min at 37° C. and 15 min at 80° C.).The destination vector (vDR243 or vDR301) was prepared by Esp3Idigestion; (1.5 μg vector was digested in 3 μl Tango Buffer, 2 I Esp3Iand water in a 30 μl reaction at 37° C. for 2 hours).

For infusion cloning, 2 μl of enhancer treated PCR product was mixedwith 100 ng Esp3I digested vector and 2 μl of 5× infusion enzyme(Clontech). The infusion reaction was done in 96-well PCR plate format.The plate was incubated for 15 min at 50° C. on a PCR machine and Stellacompetent cells were transformed by heat shock for 40 seconds at 42° C.without shaking and spread on LB agar plates with select antibiotic andincubated overnight at 37° C. Next day, colonies were picked into96-well deep well plates containing LB/Carbenicillin media and grownovernight at 37° C. Frozen stocks were made from overnight culturemixing with equal volume of 30% w/v glycerol. The V regions weresequenced using sequencing primer SPF0052. The sequences were analyzed,one positive well per hybridoma vH and vL was chosen, re-arrayed in newplates and grown overnight in rich medium with ampicillin. Clones thenhad miniprep DNA prepared for small scale transfection in 96-well plate.

Forty eight selected mouse hybridoma sequences for both heavy and lightchain were human framework adapted using an internal software program.One human framework was chosen for each one of the mouse vH or vL. Vregion DNA sequences were obtained through back-translation. SyntheticDNA regions corresponding to the FIFA amino acid sequences were orderedfrom Integrated DNA Technologies (Coralville, Iowa). Cloning wasperformed into pre-cut vDR149 and vDR157, human IgG1 and human kapparespectively. Qiagen Endo-free Maxi-prep kits were used to prepare theDNA. Expi293 (100 ml) cultures were used to express this antibody panel.

Example 5: Protocol for Human Vista-Ig T Cell Suppression Assay In Vitro

Mouse A20 cells were stably transfected with either GFP or human VISTA.They were incubated with ova peptide and with DO11.10 T cells. CD25expression by the T cells was measured 24 hours after incubation began.The A20-huVISTA cells suppress CD25 expression by the T cells, but thisreadout is significantly restored by incubation with VSTB95 (FIG. 18).

Example 6: Human Framework Regions Adaptation of Anti-Vista Antibodies

Mouse hybridoma sequences for both heavy and light chain were humanframework adapted by CDR-grafting (Jones, et al. Nature, 321: 522-525(1986) using an internal software program. The program delineates thecomplementarity determining regions (CDRs) of the V region sequencesaccording to the Kabat definitions (Wu, T. T. & Kabat, E. A. (1970). JExp Med, 132, 211-50) and compares the framework regions with the humangermline genes using Blast. The human germline with the highest sequenceidentity to the mouse frameworks was chosen as the acceptor gene forhuman framework adaptation (HFA). In a few cases, closely related humangermline genes were chosen instead, based on previous experience withwell-expressed human frameworks. DNA sequences for the human frameworkschosen for each one of the mouse vH or vL V regions were obtainedthrough back-translation. Synthetic DNA regions corresponding to the HFAamino acid sequences were ordered from Integrated DNA Technologies(Coralville, Iowa). Cloning was performed into human IgG1 and humankappa, respectively.

Example 7: Anti-Vista Antibody Constructs

Plasmid and sequence information for the molecules for cell linedevelopment: Plasmid constructs were generated for anti-VISTA antibodieshaving the VSTB112 variable regions and an IgG1κ constant regions(VSTB174, new number due to an allotypic change in the constant region),an IgG2sigma constant region (VSTB140) or an IgG1 protease-resistantconstant region (VSTB149).

Lonza Vectors

The pEE6.4 and pEE12.4 Chinese hamster ovary (CHO) expression vectorsystem (Lonza Biologics, PLC) was established in Biologics Research (BR)and Pharmaceutical Development & Manufacturing Sciences (PDMS) as theprimary expression system for generation of therapeutic mAbs inmammalian expression cell lines. Each vector contains a humancytomegalovirus (huCMV-MIE) promoter to drive the expression of theheavy chain (HC) or light chain (LC) and contains the ampicillinresistence gene. pEE12.4 vector also includes the gene encoding theglutamine synthetase (GS) enzyme. Growth conditions which requireglutamine synthetase activity places selective pressure on the cells tomaintain the expression vector (GS Gene Expression System Manual Version4.0). pEE6.4 was used to clone the HC gene and pEE12.4 to clone the LCgene as single gene vectors. The Lonza double gene plasmid is createdfrom these two Lonza single genes vectors.

Amino Acid Sequences of Variable Heavy Chain Regions of Select VISTAmAbs

>VSTB112 heavy chain  (SEQ ID NO: 37)QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSSYGWSYEFDYWGQGTLVTVSS >VSTB50 heavy chain  (SEQ ID NO: 38)QVQLVQSGSELKKPGASVKVSCKASGYTFTNYGLNWVRQAPGQGLEWMGW1NPYTGEPTYADDFKGRFVFSLDTSVSTAYLQICSLKAEDTAVYYCAREGYGNYIFPYWGQGTLVTVSS >VSTB53 heavy chain  (SEQ ID NO: 39)QVQLVQSGAEVKKPGASVKVSCKASGYTFTHYTIHWVRQAPGQGLEWMGYIIPSSGYSEYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGAYDDYYDYYAMDYWGQGTLVTVSS >VSTB95 heavy chain (SEQ ID NO: 40) EVQLVESGGGLVQPGGSLRLSCAASGFTFRNYGMSWVRQAPGKGLEWVASIISGGSYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARIYDHDGDYYAMDYWGQGTTVTVSS

Amino Acid Sequences of Variable Light Chain Regions of Select VISTAmAbs

>VSTB50 light chain  (SEQ ID NO: 41)DIVMTQTPLSLSVTPGQPASISCRASESVDTYANSLMHWYLQKPGQPPQLLIYRASNLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQTNEDPRTFGQGTKLEIK >VSTB53 light chain  (SEQ ID NO: 42)DIVMTQSPLSLPVTPGEPASISCRSSQTIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQASHVPWTFGQGTKLEIK >VSTB95 light chain  (SEQ ID NO: 43)DIVMTQSPLSLPVTPGEPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPWTFGQGTKLEIK >VSTB112 light chain  (SEQ ID NO: 44)DIQMTQSPSSLSASVGDRVTITCRASQSIDTRLNWYQQKPGKAPKLLIYSASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSAYNPITFGQGTKVEIK >VSTB116 light chain  (SEQ ID NO: 45)DIQMTQSPSSLSASVGDRVTITCRASQSINTNLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ ARDTPITFGQGTKVEIK

Example 8: ELISA and FACS Screening of Anti-Vista Antibodies

These experiments were to determine the ability of the producedantibodies to bind human or cynomolgus VISTA protein in an ELISA, aswell as to determine, using FACS screening, the ability of theantibodies to bind VISTA protein on the surface of K562 cells (humanmyelogenous leukemia cell line) expressing human or cynomolgus VISTAproteins.

Methods:

ELISA procedure summary: Plates were coated overnight at 4° C. with 1μg/ml SB0361 (human) or SB0361 (cyno (cynomolgus)) proteins, which arethe extracellular domains of VISTA from the respective species.Antibodies were diluted to 1 μg/ml as a starting concentration with 1:4step-wise dilutions for a total of 4 concentrations and incubated atroom temperature room temperature (RT) for 2 hours. Mouse anti-humanIgG1-HRP (horseradish peroxidase) was used for detection and incubatedfor 1 hour at RT. All washes were performed using PBS-Tween (0.05%).

FACS procedure summary: 2×10⁵ K562-G8 (human) or K562-C7 (cyno) cellswere stained with 5 μg/ml of each test antibody and incubated for 30minutes at 4° C. Goat anti-human IgG1-PE (phycoerythrin) antibody wasused as a secondary detection antibody at 5 μg/ml. Cells were run on aBD Fortessa and analyzed using FlowJo software (Tree Star, Inc.,Ashlang, Oreg.) for MFI (mean fluorescence intensity) of the livepopulation.

Data Analysis/Results: For each antibody, a subjective score (Yes/No)was given relating to whether the antibody bound robustly or not forboth the ELISA and FACS analysis for each of the 4 assays. If anantibody gave a “No” result for binding in either assay, it was repeatedto confirm that it was negative. The results are shown in Table 7 belowand in FIGS. 19A-19F and 20A-20F.

TABLE 7 INX Code Hu ELISA Cyno ELISA Hu FACS Cyno FACS 1 Y Y Y Y 2 Y Y YY 3 Y Y Y Y 4 Y Y Y Y 5 Y Y Y Y 6 Y Y Y Y 7 Y Y Y Y 8 Y Y Y Y 9 Y Y Y Y10 Y Y Y Y 11 N N N N 12 Y Y Y Y 14 Y Y Y Y 16 Y Y Y Y 17 Y Y Y Y 18 Y YY Y 19 Y Y Y Y 20 Y Y Y Y 21 Y Y Y Y 22 Y Y Y Y 23 N N N N 24 N N N N 25Y Y Y Y 26 N Y N Y 28 Y Y Y Y 30 N N N N 31 N N N N 32 N N N N 33 Y Y YY 34 Y Y Y Y 35 Y Y Y Y 36 Y Y Y Y 37 Y Y Y Y 38 Y Y Y Y 39 Y Y N N 40 YY Y Y 41 Y Y Y Y 42 Y Y Y Y 43 Y Y Y Y 44 Y Y Y Y 45 Y V V Y 46 Y Y Y Y47 Y Y Y Y 48 Y Y Y Y 49 Y Y Y Y

Example 9: Screening Results of Anti-Human Vista Antibodies Using theMixed Lymphocyte Reaction (Mlr) and Staphylococcus Enterotoxin B (Seb)Activation Assays

The purpose of this study was to present data supporting theidentification of multiple functional α-VISTA antibodies that enhancecellular immune responses in the mixed lymphocyte reaction (MLR) assay,as well as the staphylococcus enterotoxin B activation (SEB) assay.

The mixed lymphocyte reaction (MLR) is a standard immunological assaythat depends upon MHC class I and II mismatching to drive an allogeneicT cell response. Peripheral blood mononuclear cells are isolated fromtwo mismatched individuals, incubated together and as a result of thesemismatches, proliferation and cytokine production occurs.

Material and Methods:

10% AB Media was prepared by combining 500 ml of RPMI with 50 ml ofhuman AB serum, 5 ml of Penicillin/Streptomycin (10,000 U/ml), 5 ml ofL-glutamine (100×) and 10 ml of HEPES (1M). Media was stored for nolonger than 14 days. 1 mCi tritiated thymidine was prepared by diluting0.2 ml of thymidine stock (1 mCi/ml) in 9.8 ml of RPMI.

Soluble VISTA antibodies were diluted to 20 μg/ml in 10% AB serum media.100 μl of the appropriate antibody solutions was added to theappropriate wells of a 96 well U-bottom plate (Falcon product #353077 orequivalent). After the various cellular populations were added, thefinal concentration was 10 μg/ml.

Isolation of white blood cells: Donors were at least 18 years of age,generally healthy and selected randomly from the local population.Transferred donor blood from isolation tubes to 50 ml conicals.Under-laid 15 ml of Ficoll 1077 per 25 ml of blood being careful not tomix with the blood. Centrifuged the cells at 1250 g for 25 minutes atroom temperate with no brake. White blood cells were isolated at theinterphase of the Ficoll and the serum and diluted the cells into 40 mlof Hanks Balances Salt Solution (HBSS). Centrifuged the cells at 453 g(1500 rpm) for 10 minutes at 4° C. Resuspended the cells in 50 ml ofHBSS and counted by transferring 500 μl to a separate tube.

Mixed lymphocyte reaction (MLR) 96 well plate setup: Determined theappropriate number of “stimulator cells” and “responder cells” neededfor the assay based on the number of samples to be analyzed. Thestimulator population is seeded at 0.5×10⁵ cells/well and the responderpopulation is seeded at 1.0×10⁵ cells/well of a 96 well U-bottom plate.All conditions must be performed in triplicate. The appropriate numberof “stimulator cells” were pipetted into a new conical and centrifugedas previously described. Resuspended cells in 10 ml and irradiated with4000 rads. Centrifuged cells as previously described and resuspended ata concentration of 1×10⁶/ml in 10% AB serum media and added 50 μl toappropriate wells. Isolated the required number of responder cells andcentrifuged as previously described and resuspended at a concentrationof 2×10⁶/ml in 10% AB serum media and added 50 μl to appropriate wells.Incubated the cells for 5 days at 37° C. and 5% CO₂. On the fifth day,removed 30 μl of supernatant for analysis of interferon gamma (IFN-γ)production. On the fifth day, added 25 μl of a 40 μCi/ml tritiatedthymidine solution to each well and incubated for 8 hours at 37° C. and5% CO₂. Transferred cells to the 96 well micro scintillation plate permanufacturer's instructions. Counted using the micro scintillationcounter per manufacturer's instructions. IFN-γ concentration wasdetermined by ELISA (eBioscience cat #88-7316-88) using manufacturer'sprotocol.

Data analysis: Calculated the average counts per minute (CPM) or IFN-γconcentration for the non-treated wells. Calculated the average CPM orIFN-γ for each of the test groups. Log₁₀ transform the data set. Using12 MLR fold-scores for each compound, calculated the average for the setof 12 test groups of each compound Average score for 12 experiments=Σ[(log₁₀, (Average CPM of triplicate for test compound))−(log₁₀ (AverageCPM of triplicate for No Treatment))]/12

Acceptance criteria: All test reagents and appropriate controls weretested for endotoxin prior to running the assay and have levels of <0.1EU/mg. The responder cells alone had CPM counts below 700 CPM on averageindicating that the cells were quiescent when incubated alone. The CPMfor the MLR group was at least 2 fold higher than the CPM for respondercells incubated alone indicating that a reaction had occurred and thatthe donors are a mismatch. All MLR assays included a human IgG1 negativecontrol protein. The result of the human IgG1 negative control was notstatistically different from the non-treated samples based upon use of astudent's t-test.

Screening of anti-VISTA antibodies in the MLR: Initial screen of allcompounds. Prior to running the MLR with the anti-VISTA antibodies,antibodies were confirmed to bind both cell bound VISTA via FACSanalysis and VISTA protein via ELISA. Antibodies S26 (VSTB77), S30(VSTB86), S31 (VSTB88), S32 (VSTB90) and S39 (VSTB74) failed thisinitial screen but were still tested in the assay. For the purpose ofinitial screening, all antibodies were tested at 10 μg/ml in the MLRwith proliferation and IFN-γ being the parameters measured (FIGS.21A-21D and 22A-22B).

Selection of six lead antibodies. From the initial screen, sixcandidates were chosen for further analysis: VSTB112 (S2), VSTB116 (S5),VSTB95 (S16), VSTB50 (S41), VSTB53 (S43) and VSTB60 (S47).

Dilution studies of the top six candidates in the MLR: Protocoladjustments. The protocol is identical as previously described with theadjustment that antibodies were diluted to the following concentrations:30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01 and 0 μg/ml.

Determination of IC₅₀ values: Raw CPM counts and IFN-γ concentrationswere used to determine the IC₅₀ for each of the antibodies. Calculationsof IC₅₀ were determined through use of the program “EZ-R stats.” Sixindividual responders were used to determine the IC₅₀ values. IndividualCPM counts and IFN-γ concentrations in the MLR with dose titrations ofthe lead candidates.

TABLE 8 IC₅₀ values for both CPM and IFN-γ in the MLR VSTB112 VSTB116VSTB95 VSTB50 VSTB53 VSTB60 (S2) (S5) (S16) (S41) (S43) (S47) CPM −0.67−0.78 −0.54 −0.12 −0.33 0.02 Gamma −0.42 −0.16 0.22 0.06 0.27 0.4 **Values are in log₁₀ of antibody concentrations.

Conclusion: The initial screen indicated that multiple VISTA specificantibodies were capable of enhancing the MLR cellular immune response.Antibodies were then ranked based upon efficacy and variance and basedupon these results, VSTB112, VSTB116, VSTB95, VSTB50, VSTB53 and VSTB60were chosen to evaluate in dose-titration experiments. VSTB60 induced aweaker response than the other five antibodies in the dose-titrationexperiments.

The staphylococcus enterotoxin B (SEB) activation assay: SEB is abacterial super-antigen that induces activation of specific Vβ+ T cells.Peripheral blood mononuclear cells are isolated and incubated with theSEB antigen in culture, which induces robust cytokine production. Thisassay was conducted on the five lead candidates.

Preparation of 10% AB Media, preparation of 1 mCi tritiated thymidine,preparation of soluble VISTA antibodies, and isolation of white bloodcells were all performed as previous described above in the MLR.

SEB 96 well plate setup: Determined the appropriate number of respondercells needed for the assay based on the number of samples to beanalyzed. The responder population is seeded at 2.0×10⁵ cells/well of a96 well U-bottom plate. All conditions must were performed intriplicate. Centrifuged cells as previously described and resuspended ata concentration of 4×10⁶/ml in 10% AB serum media and added 50 μl to theappropriate wells. Added 50 μl of 10% AB serum media containing the SEBantigen at a concentration of 40 ng/ml. In the described experiments,SEB was obtained from Sigma Aldrich (cat #S0812). The finalconcentration in the well was at 10 ng/ml. Incubated the cells for 3days at 37° C. and 5% CO₂. On the third day, removed 30 μl ofsupernatant for analysis of IFN-γ production. Added 25 μl of a 1 mCi/mltritiated thymidine solution to each well and incubated for 8 hours at37° C. and 5% CO₂. Cells were transferred to the 96 well microscintillation plate per manufacturer's instructions. Counted using themicro scintillation counter per manufacturer's instructions. IFN-γconcentration was determined by ELISA (eBioscience cat #88-7316-88)using manufacturer's protocol.

Protocol: Data analysis. Calculated the average counts per minute (CPM)or IFN-γ concentration for each of antibodies at all concentrations.Acceptance criteria were performed as previously described.Determination of IC₅₀ values was performed as described. Individual CPMcounts and IFN-γ concentrations in the SEB assay with dose titrations ofthe lead candidates.

TABLE 9 IC₅₀ values for both CPM and IFN-γ in the SEB. VSTB112 VSTB116VSTB95 VSTB50 VSTB53 VSTB60 (S2) (S5) (S16) (S41) (S43) (S47) CPM −1.16−1.44 −1.12 −0.74 −1.06 not done Gamma −1.24 −0.35 0.05 1.69 −1.05 notdone **Values are in log₁₀ of antibody concentrations.

Conclusions: VISTA specific antibodies enhanced cytokine production andproliferation in a dose dependent manner in the SEB assay. IC₅₀ valuesfrom the SEB study were generally similar to the results from the MLRdilution studies.

Example 10: Epitope Binning Assay

Methods: ProteOn XPR36 system (BioRad) was used to perform epitopebinning. ProteOn GLC chips (BioRad, Cat #176-5011) were coated with twosets of 6 monoclonal antibodies (mAbs) using the manufacturerinstructions for amine-coupling chemistry (BioRad, cat #176-2410).

Competing mAbs were pre-incubated in excess (250 nM final concentration)with human VISTA (25 nM final concentration) for 4 hours at roomtemperature and 6 at a time were run over the chip coated with thepanels of coated mAbs with an association time of 4 minutes followed bydissociation for 5 minutes. Following each run, the chips wereregenerated with 100 mM phosphoric acid.

The data analysis involved grouping all sensorgrams by ligand andapplying an alignment wizard, which automatically performs an X and Yaxis alignment, and artifact removal. An Interspot correction was thenapplied to the data.

A non-competing mAb was defined as having a binding signal the sameor >A1 signal (binding to human VISTA only).

A competing mAb was defined as having binding signal <<A1 signal (i.e.,binding to human VISTA only).

Results: In the example sensorgram shown in FIG. 23, theVSTB85 antibodywas coated on the Proteon SPR chip and VISTA protein preincubated withthe indicated competitors was run over the chip. VSTB50 is an example ofa non-competitive antibody, as a positive response was seen when theVISTANSTB50 complex was run. GG8, VSTB49 and VSTB51 complexed with VISTAdid not bind to the VSTB85 coated on the chip and were thereforeclassified as competing for the same binding site on VISTA as VSTB85.

TABLE 10 Sample Set #1: coupled to sensor Sample Set #2: coupled tosensor L1 L2 L3 L4 L5 L6 L1 L2 L3 L4 L5 L6 Samples Group GG8 B85 B95B104 B112 B113 B50 B53 B66 B67 IE8 B116 GG8 1 Y Y Y Y Y Y N Y N Y Y YVSTB100.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB101.001 1 Y Y Y Y Y Y N Y N YY Y VSTB102.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB103.001 1 Y Y Y Y Y Y N YN Y Y Y VSTB104.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB105.001 1 Y Y Y Y Y YN Y N Y Y Y VSTB106.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB107.001 1 Y Y Y YY Y N Y N Y Y Y VSTB108.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB109.001 1 Y YY Y Y Y N Y N Y Y Y VSTB110.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB111.001 1Y Y Y Y Y Y N Y N Y Y Y VSTB112.001 1 Y Y Y Y Y Y N Y N Y Y YVSTB113.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB114.001 1 Y Y Y Y Y Y N Y N YY Y VSTB115.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB116.001 1 Y Y Y Y Y Y N YN Y Y Y VSTB49.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB51.001 1 Y Y Y Y Y Y NY N Y Y Y VSTB53.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB59.001 1 Y Y Y Y Y YN Y N Y Y Y VSTB65.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB67.001 1 Y Y Y Y YY N Y N Y Y Y VSTB70.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB81.001 1 Y Y Y YY Y N Y N Y Y Y VSTB92.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB95.001 1 Y Y YY Y Y N Y N Y Y Y VSTB97.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB98.001 1 Y YY Y Y Y N Y N Y Y Y VSTB99.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB50.001 2 NN N N N N Y N Y N N N VSTB54.001 2 N N N N N N Y N Y N N N VSTB56.001 2N N N N N N Y N Y N N N VSTB60.001 2 N N N N N N Y N Y N N N VSTB63.0012 N N N N N N Y N Y N N N VSTB66.001 2 N N N N N N Y N Y N N NVSTB73.001 2 N N N N N N Y N Y N N N VSTB76.001 2 N N N N N N Y N Y N NN VSTB78.001 2 N N N N N N Y N Y N N N VSTB84.001 2 N N N N N N Y N Y NN N VSTB85.001 3 Y Y Y Y Y Y N Y N Y I Y VSTB74.001 4 N N N N N N N N NN N N IE8 5 Y I Y Y Y Y N Y N Y Y Y mAb immobilized on sensor Y = Yescompeted (signal << than A1-human VISTA only) N = No competed (signal >than A1-human VISTA only) I = Inconclusive (signal similar to A1-humanVISTA only)

Example 11: Proteon Affinity Determination

Antibodies were captured on ProteOn chips using anti-IgG Fc coatedsurfaces. The antibodies were tested for binding of human and cynomolgus(cyno) VISTA extracellular domains (ECDs) at concentrations of VISTAproteins ranging from 0.39 nM to 100 nM. The antigens were allowed tobind/associate to the antibody-coated chips for 4 minutes after whichtime dissociation was monitored for 30 minutes. Chips were regeneratedwith two treatments of 100 mM phosphoric acid for 18 seconds. Allexperiments were run at 25° C. and data was fit to 1:1 Langmuir bindingmodel.

Example 12: Effects of Anti-Vista Treatment in A Mb49 Murine BladderTumor Model

Methods:

C57Bl/6 mice were injected with MB49 tumor cells. Once the tumors wereestablished, anti-VISTA treatment was initiated. Tumor growth was thenmonitored 3 times/week. Mice were euthanized, in accordance with IACUCregulations, once the tumors reached 15 mm in any dimension.

For each experiment, a frozen vial of MB49 cells was thawed and grown inRPMI 1640 (+L-Glut) with 10% serum and penicillin/streptomycinantibiotics. After three days in culture, the cells were harvested usingStemPro Accutase and resuspended in RPMI at a concentration of 5×10⁶cells/ml and 50 μl injected per mouse.

Female C57Bl/6 mice, aged 6-8 weeks were purchased from the NationalCancer Institute. Upon arrival they were allowed to acclimatize for oneday prior to having their right flanks shaved and their tails tattooed.They were then injected three-five days later.

Tumor Injection (Intradermal): Mice were injected intradermally (i.d.)on their shaved flank with 50 μl of MB49 cell suspension (˜250,000cells).

Monitoring Tumor Growth: Tumor growth was measured using electroniccalipers first across the widest dimension (L) and secondly at a 90°angle to the first measurement (W). Tumor volume derived as follows:

Volume=(L ² ·W ²)/2

Tumors were considered established once they reached ˜5 mm in diameter(˜60 mm³ volume). Once established, treatment was initiated. Tumorgrowth was measured three times per week over the course of treatmentand until the experiment was terminated.

Anti-VISTA Treatment: Chimerized 13F3-mIgG2a monoclonal antibody wasinjected intraperitoneally at 10 mg/kg. Injection schedules were thriceweekly for four weeks.

Euthanizing Mice: As per IACUC requirements, animals were euthanizedonce their tumors reached 15 mm in the longest dimension.

Analyzing Efficacy: Mouse tumor volumes were analyzed using Excel fordata management, and GraphPad Prism for graphing. Statistical analysiswas performed using a macro for R statistical computing software.

The experimental design is shown in FIG. 24.

Results:

Ch13F3-mIgG2a treatment in female mice led to complete tumor rejection(CR) in 70% of the animals and partial remission (PR) in 30% (n=7)(Table 13 and FIG. 25B). In contrast, all of the control mIgG2a-treatedmice showed progressive growth of the tumors (6/6)(FIG. 25A). These datademonstrate that anti-VISTA treatment can have a profound effect ontumor growth.

TABLE 11 Complete remission (CR) versus partial remission (PR) Female13F3 IgG2a (n = 7) CR 5 PR 2 till day 32

The human VISTA sequence is shown in FIGS. 26 and 27, adapted from Wanget al., 2011, supra, the contents of which are incorporated herein intheir entirety.

Example 13: Epitope Mapping of Anti-Vista Antibodies UsingHydrogen/Deuterium (H/D) Exchange Studies

To identify the epitopes for VSTB50, 60, 95 and 112 on human VISTA,solution hydrogen/deuterium exchange-mass spectrometry (HDX-MS) wasperformed using the corresponding Fabs. For H/D exchange, the proceduresused to analyze the Fab perturbation were similar to that describedpreviously (Hamuro et al., J. Biomol. Techniques 14:171-182, 2003; Hornet al., Biochemistry 45:8488-8498, 2006) with some modifications. Fabswere prepared from the IgGs with papain digestion and Protein A captureusing Pierce Fab Preparation Kit (Thermo Scientific, Cat #44985). Thehuman VISTA protein sequence contains six N-linked glycosylation sites.To improve the sequence coverage, the protein was deglycosylated withPNGase F. The deglycosylated VISTA protein was incubated in a deuteratedwater solution for predetermined times resulting in deuteriumincorporation at exchangeable hydrogen atoms. The deuterated VISTAprotein was in complex with either Fab of VSTB50, VSTB60, VSTB95 orVSTB112 in 46 μL deuterium oxide (D₂O) at 4° C. for 30 sec, 2 min, 10min and 60 min. The exchange reaction was quenched by low pH and theproteins were digested with pepsin. The deuterium levels at theidentified peptides were monitored from the mass shift on LC-MS. As areference control, VISTA protein was processed similarly except that itwas not in complex with the Fab molecules. Regions bound to the Fab wereinferred to be those sites relatively protected from exchange and, thus,containing a higher fraction of deuterium than the reference VISTAprotein. About 94% of the protein could be mapped to specific peptides.

The solution HDX-MS perturbation maps of VISTA with VSTB50/VSTB60, andVSTB95/VSTB112 are shown in FIG. 28 top and bottom, respectively. Twoepitope groups were identified. Anti-VISTA VSTB50 recognizes the sameepitope as VSTB60 does; VSTB95 binds to another epitope region asVSTB112 does on VISTA. Anti-VISTA VSTB50 and 60 share the same epitopewhich comprises segments, ₁₀₃NLTLLDSGL₁₁₁ (SEQ ID NO:62), and₁₃₆VQTGKDAPSNC₁₄₆ (SEQ ID NO:63) (FIG. 28 top). Anti-VISTA VSTB95 and112 appear to target similar epitopes, comprising segments₂₇PVDKGHDVTF₃₆ (SEQ ID NO:75), and ₅₄RRPIRDLTFQDL₆₅ (SEQ ID NO:65) (FIG.28 bottom). There are two other segments showing weak perturbation byVSTB95 and 112, including residues 39-52 and 118-134. However, thelevels of the reduction are not as strong as the previous regions (27-36and 54-65) in the differential map. Although one peptide, ₁₀₀TMR₁₀₂showing strong perturbation by VSTB95 and 112, is located on the otherface of VISTA surface, it is distant from the epitope regions, 27-36 and54-65. This perturbation could be due to allosteric effect. These HDX-MSresults provide the peptide level epitopes for anti-VISTA antibodies.There were no overlapping epitope regions for these two epitope groups.These results are in agreement with the previous competition binningdata in that they do not compete with each other.

Example 14: Structure Determination of the Human Vista ECD:VSTB112 FabComplex by Protein Crystallography

In an effort to determine the VISTA structure and to delineate theepitope and paratope defining the interaction between VISTAextracellular domain (ECD) and the Fab fragment of lead antibodyVSTB112, the complex was crystallized and structure determined to 1.85 Åresolution. The structure of the ECD of human VISTA in complex with theFab fragment of the antibody VSTB112 was determined in an effort both todetermine the structure of VISTA ECD itself and to define theepitope/paratope for this interaction. The structure reveals VISTA toadopt an IgV fold with a chain topology similar to the TCR Vα chain. Inaddition to the canonical disulfide bond bridging B and F strands in theback and front faces of the β-sandwich, the structure reveals the ECD tohave two additional disulfide bonds, one tethering the CC′ loop to thefront sheet and a second between the A′ and G′ strands. Although crystalcontacts between VISTA molecules are present, they are minor and thereis no evidence for a dimer of VISTA ECDs based on this structure. TheVSTB112 epitope is shown to comprise the portions of the VISTA BC, CC′,and FG loops together with residues of the front beta sheet (C′CFG)nearest those loops. The paratope is biased largely toward heavy chaininteractions with CDR L3 making minimal contact.

Epitope/Paratope Defining VISTA:VSTB112 Interaction

VSTB112 Fab buries a surface area of 1024.3 Å2 upon binding VISTA ECD,with burial of the heavy chain surface accounting for 715.3 Å2 of thistotal. Seven hydrogen bonds and 4 salt bridge interactions are formedbetween VISTA and VSTB112 light chain and 10 hydrogens and 2 salt bridgeinteractions between VISTA and VSTB112 heavy chain. VSTB112 recognizesresidues in the front sheet strands C′, C, F, and G on the ends proximalto the FG loop as well as residues in the BC, FG, and CC′ loops (FIGS.29 and 30). Interactions with the CC′ loop account for most of thecontacts with the Fab light chain with only residues E125 and R127 inthe FG loop making additional light chain interactions. Residues 119 to127 corresponding to the VISTA FG loop account for 38% of the total1034.8 Å2 of surface area buried upon binding VSTB112. Notably, thisloop is highly polar, comprised of the following sequence-IRHHHSEHR-(SEQ ID NO:76). Additionally, W103 in the VSTB112 CDR H3 packs nicelyagainst the backbone of VISTA residues H122 and H123, and VISTA H121makes an edge on interaction with the aromatic ring of F55 in CDR H2.

A comparison of epitope regions identified by crystallography and HDX isshown in FIG. 31.

Example 15: Activation of T Cells and Monocytes by Anti-Vista Antibodies

The functional effect of anti-VISTA antibodies was evaluated in two invitro assays, mixed leukocyte reaction (MLR) and SEB (Staphylococcusenterotoxin B). Both assays measure T cell proliferation and cytokineinduction as their primary readouts, but these effects are due todifferent mechanisms. In the MLR, peripheral blood mononuclear cells(PBMCs) from two different human donors are incubated together, andmajor histocompatibility complex (MHC) mismatch between T cells of onedonor and dendritic cells of the other donor results in T cellproliferation and interferon (IFNγ) production. In the SEB assay, PBMCsfrom a single donor are incubated with a bacterial superantigen, whichdirectly links MHC Class II protein on the surface of antigen-presentingcells (APC) to the T-cell receptor (TCR) on T cells, causing T cellactivation, proliferation, and cytokine secretion. In both assays,VSTB112, which is the parent molecule of VSTB174, demonstrateddose-dependent induction of T cell proliferation and cytokineproduction, and was most potent among the candidates (FIGS. 21A-21D,Table 12).

TABLE 12 EC50 values for the MLR assay readouts. VSTB112 (parent ofVSTB174) was the most potent molecule. EC₅₀ EC₅₀ IFNγ proliferationproduction Candidate (μg/ml) (μg/ml) VSTB112 0.21 0.38 VSTB116 0.17 0.69VSTB95 0.29 1.67 VSTB50 0.77 1.14 VSTB53 0.47 1.88 VSTB60 1.04 2.48

Monocyte Activation Assays

The assay data, shown in Table 12, was generated with VSTB112, theparent molecule of VSTB174. To better understand the activity ofVSTB174, monocyte activation assays were conducted. The results showedthat incubation of VSTB174 with whole PBMCs induced upregulation ofactivation markers (CD80 and HLA-DR) on CD14+ monocytes, indicating aneffect of antibody binding to an immune cell subset known to expresshigh levels of VISTA (FIG. 32). A further question is whether theeffects on monocyte activation in whole PBMC could be facilitated by anyantibody that binds VISTA and has an IgG1 Fc. Antibodies VSTB103 andVSTB63 bind to VISTA with high affinity (KD 6.36E-10 and 8.30E-10respectively) and to cells expressing VISTA protein, similar to VSTB112and VSTB111. VSTB103 is in the same epitope bin as VSTB112, while VSTB63is in a different epitope bin; neither antibody facilitated monocyteactivation. Taken together, these results show that one mechanism bywhich VSTB174 may exert its effect on T cell activation/proliferation isvia monocyte activation facilitated by NK cells.

Preparation of Media

500 ml of RPMI 1640 (Corning, 10-040-CV) was combined with 50 ml ofhuman AB serum (Valley Biomedical, Inc, Lot #3C0405), 5 ml ofPenicillin/Streptomycin (Lonza, 17-602E) 10,000 U/ml, 5 ml ofL-glutamine (100×) (Gibco, 25030-081) and 10 ml of HEPES (1M) (FisherBP299-100, Lot #-1). Media was stored for no longer than 14 days at 4°C.

Preparation of Soluble VISTA and Control Antibodies

Antibodies were diluted to 2× desired concentration in 10% AB serummedia: VSTB174: lot VSTB174.003

Added 100 μl of the appropriate antibody solutions to the appropriatewells of a 96 well U-bottom plate (Falcon, 353077). After the variouscellular populations were added in 100 μl the final concentration ofeach antibody was 1, 0.1 or 0.01 g/ml. IgG1 control antibody CNTO 3930(Lot 6405, ENDO <0.1 EU/mg) was added at a final concentration of 1μg/ml.

The PBMCs were isolated

Donors were at least 18 years of age, generally healthy and selectedrandomly from the local population.

Donor blood was transferred from isolation tube to 50 ml conicals.

15 mls of Ficoll 1077 (SIGMA, 10771) were under-laid being careful notto mix with the blood. This was per 25 mls of blood.

The cells were centrifuged at 1250 g for 25 minutes at room temperaturewith no brake.

The white blood cells were isolated at the interphase of the Ficoll andthe serum and the cells were diluted into 40 ml of Hanks Balanced SaltSolution (HBSS).

The cells were centrifuged at 453 g (1500 rpm) for 10 minutes at 4 C.

The cells were resuspended in 50 mls of HBSS and were counted bytransferring 500 l to a separate eppendorf tube.

Additionally, a Pan Monocyte isolation kit from Miltenyi was used permanufacturer's instructions (cat #130-096-537) to isolate CD14+ cells bynegative selection in several treatment groups.

In Vitro Culture Setup

The appropriate number of cells needed was determined for the assaybased on the number of samples to be analyzed. The responder populationwas seeded at 2.0×10⁵ cells/well of a 96-well U-bottom plate. For theCD14 negatively selected population, 0.5×10⁵ cells were seeded. Allconditions were performed in triplicate.

The cells were centrifuged as described above and resuspended at aconcentration of 2×10⁶/ml for the whole PBMC population and 0.5×10⁶/mlfor the CD14 negatively selected population in 10% AB serum media andadded 100 l of test antibody to appropriate wells bringing the totalvolume in each well to 200 l.

The cells were incubated for 1, 2, or 3 days at 37° C. and 5% CO₂.

Antibody Staining and Flow Cytometry

The 96 well U-bottom plate was centrifuged for 5 minutes at 453 g andremoved the supernatant.

Cells were washed with 200 μl PBS and centrifuged as in step 5.5.1.

The supernatant was discarded and resuspended in 50 μl of PBS containingthe following antibodies:

-   -   CD14-APC (clone HCD14) 1:250 (Biolegend cat #325608)    -   HLA-DR-PE Cy7 (clone L243) 1:250 (Biolegend cat #307616)    -   CD80-PE (clone 2D10) 1:250 (Biolegend cat #305208)    -   Hu FcR binding inhibitor (eBioscience cat #14-9161-73)

Was incubated for 20 minutes on wet ice in the dark.

150 μl of PBS was added and centrifuged as in step 5.5.1.

150 l of PBS buffer was added and analyzed via FACS.

Samples were run on a Miltenyi MACSQuant 10-parameter flow cytometer andanalyzed using FlowJo 9.7.5 for expression of HLA-DR and CD80 on theCD14+ population. Geometric mean fluorescence intensity (MFI), astatistic that defines the central tendency of a set of numbers, wasused as the defining statistic to compare treatments.

Statistical Analysis

All statistics were carried out in Prism GraphPad, version 6. Pair-wisecomparisons amongst the groups were made at each of the time-pointsusing One-Way ANOVA with Tukey correction for multiplicity. P-valuesless than 0.05 for all tests and comparisons were deemed significant.For all graphs and tables, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Example 16: ADCC and ADCP Activities of Anti-Vista Antibodies

VSTB174 has an IgG1 Fc, which can confer antibody-dependentcell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediatedphagocytosis (ADCP) activity. Both types of assays were conducted andshowed that VSTB174 could lyse or phagocytose K562-VISTA cells (FIGS.33-34), but not K562 myeloma cell line parental cells (data not shown).An additional mechanism of action of VSTB174 to modulate the inhibitoryaction of VISTA may be the lysis or engulfment of cells expressing highlevels of VISTA, thus removing them from the local microenvironment.

Example 17: ADCP Activities of Additional Anti-Vista Antibodies

An in vitro phagocytosis assay was used to study the enhancement ofmacrophage-mediated phagocytosis of cells ectopically expressing VISTAby anti-human VISTA mAbs (VSTB173 and VSTB174). These mAbs were clonedinto different Fc backbones (IgG1 WT (wild type), IgG1 PR (proteaseresistant), and IgG2a) and were postulated to potentially have differentactivities with respect to enhancing phagocytosis. The IgG1 and IgG1 PRbackbones are capable of binding to Fe receptors and have the potentialto cause ADCP, while the IgG2a does not bind to Fc receptors and shouldnot mediate ADCP.

Anti-VISTA antibodies were tested in ADCP assays with K562 parental andK562-VISTA target cells. As shown in FIGS. 35-36, VSTB174, VSTB149,VSTB173 and VSTB145 enhanced hMac phagocytosis of K562-VISTA cells.VISTA antibodies VSTB140 or VSTB132, with the IgG2σ Fc that did not bindFc receptors, did not enhance phagocytosis as expected. VISTA mAbsVSTB174 and VSTB173 with IgG1 Fc showed more robust phagocytosis thanVSTB149 and VSTB145 with the IgG1PR Fc (see Tables 13 and 14 for EC₅₀values).

TABLE 13 Anti-human VISTA mAb EC₅₀ values. Treatment VSTB174 VSTB149VSTB140 EC₅₀ 0.0782 0.1142 NA

TABLE 14 Anti-human VISTA mAb EC₅₀ values. Treatment VSTB173 VSTB145VSTB132 EC₅₀ 0.0146 0.1075 NA

VSTB174 and VSTB173 showed weak enhancement of phagocytosis of K562parental cells at the highest concentration (FIGS. 35-36), which may bedue to low expression of VISTA by the K562 cells. The other anti-VISTAantibodies did not enhance phagocytosis of the K562 cells.

The negative control antibodies were each tested at two differentconcentrations in the K562-VISTA phagocytosis assay, but did not induceany phagocytosis. This result indicates that the phagocytosis mediatedby the anti-VISTA antibodies is specific and due to VISTA antigenexpression by the K562-VISTA cells.

Example 18: ADCC Activities of Additional Anti-Vista Antibodies

In order to test their ability to induce ADCC, the following three humananti-VISTA antibodies were tested:

VSTB174 (IgG1)

VSTB149 (IgG1 PR)

VSTB174.LF (IgG1 LF (low fucose)).

Each antibody was tested at six different concentrations within the sameplate, in triplicate over two separate experiments for a total of sixdata points.

VSTB174, VSTB149, and VSTB174.LF each demonstrated measurable ADCCactivity at 10, 1, 0.1 and 0.01 μg/mL, while only the LF antibodydemonstrated measurable ADCC activity at 0.001 μg/mL; none of theantibodies demonstrated ADCC at 0.0001 μg/mL. As each of theseantibodies has an IgG1 or IgG1 variant Fc, this result is expected. TheLF antibody demonstrated increased ADCC potency as evidenced by thesmaller EC₅₀ value for the LF antibody curve (0.002293 μg/mL) ascompared to the regular IgG1 antibody curve (0.02381 μg/mL). The IgG1 PRantibody curve had an EC₅₀ value similar to the regular IgG1 curve(0.01846 μg/mL).

TABLE 15 EC₅₀ values (μg/mL) of three tested anti-VISTA antibodies asdetermined by ADCC analysis. anti-VISTA Antibody EC₅₀ (μg/mL) VSTB174(IgG1) 0.02381 VSTB149 (IgG1 PR) 0.01846 VSTB174.LF (IgG1 LF) 0.002293

The human IgG1, human IgG1 PR and human IgG1 LF antibodies all showedmeasurable ADCC mediated killing at the 10, 1, 0.1 and 0.01 μg/mLantibody concentrations, while only the LF antibody showed killing atthe 0.001 μg/mL antibody concentration. None of the anti-VISTAantibodies showed killing at the 0.0001 μg/mL antibody concentration.

The LF antibody showed approximately 10 times more potent ADCC killingthan either the regular IgG1 antibody or the IgG1 PR antibody, as seenin the EC50 values.

Example 19: Affinity of VSTB174 for Human and Cynomolgus Vista

The affinity of VSTB174 for human and cynomolgus monkey VISTAextracellular domain (ECD) was determined by surface plasmon resonance(SPR) methods on a ProteOn instrument. VSTB174 displayed very similar KDvalues for each protein, 1.56E-10 M for human VISTA ECD and 8.66E-11 Mfor cynomolgus VISTA.

Example 20: Vista Antibodies Exhibit Efficacy in Murine Tumor Models

Mouse Strains, Reagents and Tumor Models

For the in vivo studies, human VISTA knockin (VISTA-KI) miceback-crossed onto a C57Bl/6 background were used.

An anti-human VISTA antibody was generated to enable testing in theVISTA-KI mice, using the VSTB174 variable region grafted onto mouse FcIgG2a (VSTB123).

The MB49 bladder cancer was evaluated in the VISTA KI mice,

In addition to published studies demonstrating that anti-VISTA antibodytherapy inhibits tumor growth in wild type mice (Le Mercier et al.,2014), anti-tumor efficacy has been demonstrated with the surrogatehamster antibody in wt mice using different dosing schedules, and in theVISTA-KI mice treated with VSTB123.

In Vivo Efficacy Studies in the MB49 Tumor Model in VISTA-KI Mice

MB49 efficacy studies were conducted in female VISTA-KI mice, testingVSTB123 at several doses ranging from 1-10 mg/kg. Mice were injectedintradermally with 250,000 MB49 tumor cells on day 0. On day 6, dosingbegan as indicated in FIG. 37 (either 10 mg/kg of the isotype controlmIgG2a, or the indicated doses of VSTB123; 10 mice/group).

VSTB123 was more effective at higher vs lower doses, as shown in FIG.37. Doses of 10 mg/kg and 7.5 mg/kg were equivalent, while tumors grewmore quickly in the mice dosed at 5 or 1 mg/kg.

Example 21: Detection of Vista Expression in Human Tumors withAnti-Vista Antibodies

FIG. 1 shows VISTA expression by an AML tumor cell line—this and the RNAseq expression data in FIG. 17 support the idea that VISTA is expressedby AML cells and that anti-VISTA drug be efficacious through directlytargeting these cells for immune modulation or antibody-mediatedkilling.

Data to evaluate VISTA expression in lung cancer was obtained from lungtumor samples from surgical resections. Cells were dissociated andcharacterized for expression of VISTA and many other markers. Resultsshowed that 13/13 lung tumors (squamous or adenocarcinomas) containedCD14+ VISTA+ myeloid cells, (FIG. 38).

Example 22: Detection of Vista Expression in Lung Tumors UsingAnti-Vista Antibodies

An immunohistochemistry assay was developed using clone GG8, ananti-human VISTA mouse IgG1. This mAb was used to investigate thestaining of VISTA in non small cell lung cancer (NSCLC) FFPE tumorsections.

FFPE tumor sections were treated with standard antigen retrieval methodsprior to staining. GG8 mouse anti-human VISTA antibody was used at a1:500 dilution. GG8 binding was detected using a rabbit anti-mousepolyclonal antibody, followed by anti-rabbit polymer HRP. Counterstainwith hematoxylin followed, then tumor sections were scored.

VISTA expression in lung cancer was mostly restricted to the immuneinfiltrate (example shown in FIG. 39) and high levels of VISTA positivecells were present in many lung cancer samples

Example 23: Structure of the Extracellular Domain (Ecd) of Human Vistain Complex with the Fab Fragment of VSTB174

VISTA antigen variants were generated and purified for crystallography.Recombinant his-tagged VSTB174 Fab was internally expressed andpurified. Crystals were generated and used to collect higher resolutiondata for the VISTA ECD:VSTB174 Fab complex using synchrotron radiationand the structural determination was solved using combinations ofhomology modeling and electron density analyses (FIG. 29(Top)).

The structure of the VISTA ECD:VSTB174 Fab complex was determined byx-ray crystallography to a resolution of 1.85 Å, providing the firststructure of the VISTA ECD and delineating the VSTB174 epitope andparatope. The VISTA ECD adopts an IgV fold with a topology similar toCTLA-4 ECD, but possesses a unique G′ strand that extends the frontsheet of the β-sandwich. A′ and G′ are further tethered chemically via adisulfide bridge formed between residues C12 in the A′ strand and C146in the G′ strand. Six cysteines were found to be engaged in threeintramolecular disulfide bonds, and, based on crystal contacts, there isno evidence for a dimeric VISTA.

VSTB174 recognizes residues in the front sheet strands C′, C, F, and Gon the ends proximal to the FG loop as well as residues in the BC, FG,and CC′ loops.

The teachings of all patents, published applications and referencescited herein are incorporated by reference in their entirety.

While this invention has been particularly shown and described withreferences to example embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1-103. (canceled)
 104. An isolated or recombinant nucleic acid whichencodes for an antibody or antibody fragment thereof comprising anantigen binding region that binds to a V-domain Ig Suppressor of T cellActivation (VISTA), which antibody: (i) comprises a VH domain comprisinga VII CDR1 having the amino acid sequence of SEQ ID NO: 25, a VH CDR2having the amino acid sequence of SEQ ID NO:26 and a VH CDR3 having theamino acid sequence of SEQ ID NO:27, and (ii) comprises a VL domaincomprising a VL CDR1 having the amino acid sequence of SEQ ID NO:28, aVL CDR2 having the amino acid sequence of SEQ ID NO: 29 and a VL CDR3having the amino acid sequence of SEQ ID NO:
 30. 105. The nucleic acidof claim 104, wherein the encoded antibody or antibody fragmentcomprises antibody or antibody fragment is a Fab, F(ab′)₂, or scFvantibody fragment.
 106. The nucleic acid of claim 104, wherein theencoded antibody or antibody fragment is a human or humanized antibody.107. The nucleic acid of claim 104, wherein the encoded antibody orantibody fragment comprises a variable heavy chain polypeptide having asequence at least 90% identical to SEQ ID NO:37.
 108. The nucleic acidof claim 104, wherein the encoded antibody or antibody fragmentcomprises a variable heavy chain polypeptide having a sequence at least95% identical to SEQ ID NO:37.
 109. The nucleic acid of claim 104,wherein the encoded antibody or antibody fragment comprises a variableheavy chain polypeptide identical to SEQ ID NO:37.
 110. The nucleic acidof claim 104, wherein the encoded antibody or antibody fragmentcomprises a variable light chain polypeptide having a sequence at least90% identical to SEQ ID NO:
 44. 111. The nucleic acid of claim 104,wherein the encoded antibody or antibody fragment comprises a variablelight chain polypeptide having a sequence at least 95% identical to SEQID NO:44.
 112. The nucleic acid of claim 104, wherein the encodedantibody or antibody fragment comprises a variable light chainpolypeptide identical to SEQ ID NO:44.
 113. The nucleic acid of claim104, wherein the encoded antibody or antibody fragment comprises avariable light chain polypeptide having a sequence at least 90%identical to SEQ ID NO:44 and a variable heavy chain polypeptide havinga sequence at least 90% identical to SEQ ID NO:37.
 114. The nucleic acidof claim 104, wherein the encoded antibody or antibody fragmentcomprises a variable light chain polypeptide having a sequence at least95% identical to SEQ ID NO:44 and a variable heavy chain polypeptidehaving a sequence at least 95% identical to SEQ ID NO:37.
 115. Thenucleic acid of claim 104, wherein the encoded antibody or antibodyfragment comprises a variable light chain polypeptide having a sequenceidentical to SEQ ID NO:44 and a variable heavy chain polypeptideidentical to SEQ ID NO:37.
 116. The nucleic acid of claim 104, whereinthe encoded antibody comprises a human constant region.
 117. The nucleicacid of claim 104, wherein the encoded antibody comprises a human IgG1constant domain.
 118. The nucleic acid of claim 114, wherein the encodedhuman constant domain comprises a mutation which impairs FcR binding orprotease cleavage.
 119. The nucleic acid of claim 104, wherein theencoded antibody comprises a human IgG1 heavy constant domain having thehuman IgG1 heavy constant domain sequence comprised m SEQ ID NO:55, SEQID NO:59, SEQ ID NO:60 or SEQ ID NO:61.
 120. The nucleic acid of claim104, which comprises a human IgG1 light constant domain having the humanIgG1 light constant domain sequence comprised in SEQ ID NO:56.
 121. Thenucleic acid of claim 104, wherein the encoded antibody comprises ahuman IgG1 light constant domain having the human IgG1 light constantdomain sequence comprised in SEQ ID NO:56.
 122. The nucleic acid ofclaim 104, wherein the encoded antibody comprises the heavy chainpolypeptide of SEQ ID NO:55, 59, 60 or 61 and the light chainpolypeptide of SEQ ID NO:
 56. 123. The nucleic acid of claim 104,wherein the encoded antibody comprises the heavy chain polypeptide ofSEQ ID NO: 61 and the light chain polypeptide of SEQ ID NO:
 56. 124. Avector comprising the nucleic acid of claim
 104. 125. A vectorcomprising the nucleic acid of claim
 115. 126. A vector comprising thenucleic acid of claim
 122. 127. A vector comprising the nucleic acid ofclaim
 123. 128. A recombinant cell which comprises a nucleic acidaccording to claim 104, operably linked to a promoter.
 129. Arecombinant cell which comprises a nucleic acid according to claim 115,operably linked to a promoter.
 130. A recombinant cell which comprises anucleic acid according to claim 122, operably linked to a promoter. 131.A recombinant cell which comprises a nucleic acid according to claim123, operably linked to a promoter.
 132. A recombinant cell according toclaim 128, which comprises a mammalian cell.
 133. A recombinant cellaccording to claim 131, which comprises a mammalian cell.
 134. Arecombinant cell according to claim 128, which comprises a CHO cell.135. A recombinant cell according to claim 131, which comprises a CHOcell.
 136. A method of producing an antibody or antibody fragmentencoded by the nucleic acid of claim 104 comprising culturing anisolated or recombinant cell comprising said nucleic acid operablylinked to a promoter under conditions that result in the expression ofsaid antibody or antibody fragment.
 137. A method of producing anantibody or antibody fragment encoded by the nucleic acid of claim 115comprising culturing an isolated or recombinant cell comprising saidnucleic acid operably linked to a promoter under conditions that resultin the expression of said antibody or antibody fragment.
 138. A methodof producing an antibody or antibody fragment encoded by the nucleicacid of claim 122 comprising culturing an isolated or recombinant cellcomprising said nucleic acid operably linked to a promoter underconditions that result in the expression of said antibody or antibodyfragment.
 134. A method of producing an antibody or antibody fragmentencoded by the nucleic acid of claim 122 comprising culturing anisolated or recombinant cell comprising said nucleic acid operablylinked to a promoter under conditions that result in the expression ofsaid antibody or antibody fragment.